Analog of Haemophilus Hin47 with reduced protease activity

ABSTRACT

An isolated and purified analog of Haemophilus influenzae Hin47 protein has a decreased protease activity which is less than about 10% of that of natural Hin47 protein and preferably substantially the same immunogenic properties as natural Hin47 protein. An isolated an purified nucleic acid module encoding the Hin47 analog may be provided in a recombinant plasmid which may be introduced into a cell which is grown to produce the Hin47 analog. Immunogenic compositions comprises the Hin47 analog and the encoding nucleic acid may be formulated as vaccines for in vivo administration to a host, including a human, to confer protection against diseases caused by a bacterial pathogen, including Haemophilus species, such as Haemophilus influenzae, that produces Hin47 protein or a protein capable of inducing antibodies in the host specifically reactive with Hin47 protein. The Hin47 analog and the encoding nucleic acid also may employed in diagnostic applications.

REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 08/615,271, filed Jun. 20, 1996 (now U.S. Pat. No. 5,981,503) which itself, by virtue of being a U.S. National Phase Application under 35 USC 371 of PCT/CA95/00434 filed Jul. 21, 1995, is a continuation of U.S. patent application Ser. No. 08/487,167, filed Jun. 7, 1995 (now U.S. Pat. No. 5,869,302) which itself is a continuation-in-part of U.S. patent application Ser. No. 08/296,149, filed Aug. 26, 1994 (now U.S. Pat. No. 5,939,297), which itself is a continuation-in-part of U.S. patent application Ser. No. 08/278,091, filed Jul. 21, 1994 (now U.S. Pat. No. 5,506,139).

FIELD OF THE INVENTION

The present invention relates to the field of immunology and is particularly concerned with immunogens and antigens from species of Haemophilus.

BACKGROUND OF THE INVENTION

Haemophilus influenzae is the organism responsible for a variety of serious human diseases, such as meningitis, epiglotitis, pneumonia and otitis media. Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis in children under the age of five years. Protective antibodies to the disease are induced by the capsular polysaccharide of the organism and vaccines have been developed that utilise the purified polyribosyl ribitol phosphate (PRP) is the antigen. This vaccine provides 90% protection in adults and in children over 24 months of age, but was ineffective in children under 24 months (Zangwill et al 1993). (The references are identified in a list of references at the end of this disclosure, each of which reference in the list is hereby incorporated by reference without further reference thereto). Like other polysaccharide antigens, PRP does not induce the proliferation of T-helper cells, and reimmunisation fails to elicit either a booster response or an increase in memory cells. Conjugation of the PRP polysaccharide with protein carriers confers T-cell dependent characteristics to the vaccine and substantially enhances the immunolgic response to the PRP antigen. Currently, there are four PRP-carrier conjugate vaccines available. These are vaccines based upon H. influenzae type b capsular polysaccharide conjugated to diphteria toxoid, tetanus toxoid, or Neisseria meningitidis outer membrane protein (reviewed in Zangwill et al., 1993). These H. influenzae b conjugate vaccines have dramatically reduced the incidence of bacterial meningitis (Schoendorf et al, 1994).

There are six serotypes of H. influenzae designated a to f, which are defined by their capsular polysaccharides. The current Haemophilus conjugate vaccines do not protect against other invasive typable strains (types a and c) and, importantly, do not protect against non-typable (NTHi) strains which are a common cause of postpartum and neonatal sepsis, pnemonia and otitis media. Otitis media is the most common illness of early childhood with approximately 70% of all children suffering at least one bout of otitis media before the age of seven. Chronic otitis media can lead to hearing, speech, and cognitive impairment in children. It is caused by bacterial infection with Streptococcus pneumoniae (approximately 50%), non-typable H. influenzae (approximately 30%), and Moraxella (Branhamella) catarrhalis (approximately 20%). In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. To achieve universal protection against H. influenzae related diseases, particularly in the two to six month age group and certain high risk groups, the provision of conserved, cross-reactive non-capsular H. influenzae immunogens is desirable. Non-typable strains of H. influenzae are also important pathogens responsible for pneumonia in the elderly and other individuals who are particularly susceptible to respiratory infections. There is thus a need for antigens from H. influenzae which are useful as components in immunogenic preparations that provide protection against the many serotypes of H. influenzae. PCT application WO 92/10936, publisehd Jul. 9, 1992 and incorporated herein by reference thereto, describes a 47,000 molecular weight outer membrane protein obtained from H. influenzae that is reported to be an adhesin and has been termed Hin47 that is immunologically conserved between non-typable, type b and non-typed clinical isolates of H. influenzae. The amino acid sequence of Hin47 and the nucleotide sequence of the gene encoding Hin47 were presented at the American Society of Microbiology (ASM) conference held in New Orleans, May 26-30, 1992. These sequence have also been published in PCT application WO 94/00149, published Jan. 6, 1994 and incorporated herein by reference thereto.

Since Hin47 is conserved among strains of Haemophilus influenzae, and is reported to be an adhesin, the protein has utility in diagnosis of and vaccination against disease caused by H. influenzae or other bacterial pathogens that produce Hin47 or a protein capable of raising antibodies specifically reactive with Hin47.

A disadvantage of Hin47 for use as an antigen in diagnosis, for the generation of anti-Hin47 antibodies useful in diagnosis and as an immunogen in vaccination is the unexpected discovery by the present applicants that Hin47 has protease activity which results in the autodigestion of Hin47 and the proteolytic degradation of other antigens mixed therewith.

It would be advantageous to provide analogs of Hin47 protein (sometimes referred to herein as mutants or derivatives) that are substantially reduced in proteolytic activity for use as antigens, immunogenic preparations including vaccines, carriers for other immunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of analogs of Haemophilus Hin47 protein having reduced protease activity.

In accordance with one aspect of the invention there is provided an isolated and purified analog of Haemophilus influenzae Hin47 protein having a decreased protease activity which is less than about 10% of natural Hin47 protein. Such Hin47 analog preferably has substantially the same immunogenic properties of natural Hin47 protein. The analog of the present invention may be produced by chemica, biochemical or genetic modification of natural Hin47.

In one embodiment of the present invention, when the analog is produced by genetic modification, at least one amino acid of the natural Hin47 contributing to protease activity may be deleted or replaced by a different amino acid to produce the reduced protease activity. Alternatively, the reduced protease activity may be achieved by inserting at least one amino acid into the natural Hin47 protein. The at least one deleted or replaced amino acid may be selected from amino acids 195 to 201 of Hin47, and specifically may be Serine-197, which may be deleted or replaced by alanine, cysteine or threonine. In addition, the at least one deleted or replaced amino acid may be His-91 and may be deleted or replaced alanine, lysine or arginine. Further, the at least one deleted or replaced amino acid may be Asp-121 and may be deleted or replaced by alanine.

In addition, multiple amino acids in the Hin47 molecule may be deleted or replaced. Such multiple amino acids may include His-91 and Serine-197 and may be deleted or replaced by Ala-91 and Ala-197 to produce a Hin47 analogue H91A/S197A. In addition, the multiple amino acids may include His-91, Asp121 and Ser-197 and may be deleted or replaced with Ala-91, Ala-121 and Ala-197 respectively to produce a Hin47 analogue H91A/D121A/S197A. A summary of some of the properties of some Hin47 analogues as provided herein is shown in Table 3. Only one Hin47 mutant D121E was found to retain substantial protease activity.

In a further aspect, the present invention provides an isolated and purified nucleic acid molecule comprising a mutant Haemophilus influenzae hin47 gene encoding an analog of Haemophilus influenzae Hin47 protein having a reduced protease activity which is less than about 10% of natural Hin47 protein. The mutant hin47 gene may encode any of the Hin47 analogs discussed above. The mutant gene preferably is formed by site-directed mutagenesis of a wild-type hin47 gene. The nucleic acid molecule may be contained in a recombinant plasmid adapted for transformation of a host and may be plasmid DS-1011-1-1 (deposited on Jul. 27, 1994 at American type Culture Collection, Rockville, Md., U.S.A. under Accession No. 75845). The invention also includes a transformed cell containing such a recombinant plasmid.

The present invention, in another aspect, includes a method for producing an analog of Haemophilus influenzae Hin47 protein having a reduced protease activity which is less than about 10% of natural Hin47 protein, which comprises identifying at least one amino acid residue of Hin47 protein which contributes to protease activity thereof, effecting site-directed mutagenesis of the hin47 gene to remove or replace a nucleotide sequence encoding the at least one amino acid and to produce a mutated hin47 gene, introducing the mutated hin47 gene into a cell to produce a transformed cell and growing the transformed cell to produce the Hin47 analog. The at least one amino acid which is selected may be any of the ones specifically identified above with respect to the Hin47 analog.

The introduction of the mutated hin47 gene preferably produces a transformed cell in which the mutated hin47 gene is under control of the T7 promoter and the growing of the transformed cell and expression of the Hin47 analog by the T7 promoter then preferably is effected by culturing in an inducing concentration of lactose. Preferably, the introduction of the mutated hin47 is effected by transforming the cell with the recombinant plasmid DS-1011-1-1, sometimes otherwise referred to as plasmid pT7/Hin47*.

A further aspect of the invention provides a method of providing isolated and purified Hin47 analog, which comprises effecting the procedure described above for the production of the Hin47 analog to produce grown transformed cells harbouring inclusion bodies containing the Hin47 analog, disrupting the grown transformed cells to produce supernatant and the inclusion bodies, solubilizing the inclusion bodies to produce a solution containing Hin47 analog, chromatographically purifying the Hin47 analog from the solution free from cell debris, and isolating the purified Hin47 analog.

The analogs of Hin47 provided herein with their decreased proteolytic activity are useful as antigens in immunogenic composition, carriers for other immunogens, diagnostic agents and in the generation of diagnostic agents. The nucleic acid molecules also are useful as probes for diagnostic use and also as in immunogenic compositions.

In a further aspect of the invention, there is provided an immunogenic composition comprising an immunoeffective amount of the Hin47 analog or of the nucleic acid molecule including the gene encoding the Hin47 analog. The immunogenic composition may be formulated as a vaccine for in vivo administration to a host, including a human, to confer protection against diseases caused by a bacterial pathogen that produces Hin47 or a protein capable of inducing antibodies in the host specifically reactive with Hin47. The bacterial pathogen may be a Haemophilus species, such as Haemophilus influenzae. The immunogenic compositions of the invention may further comprise at least one other immunogenic or immunostimulating material, such as an adjuvant. In an additional embodiment, the nucleic acid molecule comprising a gene encoding the Hin47 analog may be contained with a live vector, such as a pox virus, Salmonella, poliovirus, adenovirus, vaccinia or BCG.

The invention also extends to a method of generating an immune response in a host, including a human, comprising administering thereto an immuno-effective amount of the immunogenic compositions provided herein.

As mentioned above, the Hin47 analog provided herein is useful in diagnostic applications. Accordingly, in an additional aspect of the invention, there is provided a method of determining the presence of antibodies specifically reactive with Hin47 in a sample, comprising the steps of:

(a) contacting the sample with the Hin47 analog having substantially the same immunogenic properties as the natural Hin47 protein as provided herein to produce complexes comprising the Hin47 analog and any such antibodies present in the sample specifically reactive therewith; and

(b) determining production of the complexes.

The present invention also provides a method of determining the presence of Hin47 in a sample, comprising the steps of:

(a) immunizing a subject with an immunogenic composition as provided herein to produce antibodies specific for Hin47 protein;

(b) contacting the sample with the antibodies to produce complexes comprising any Hin47 present in the sample and the Hin47 specific antibodies; and

(c) determining production of the complexes.

The invention also extends to a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with Hin47, comprising:

(a) the Hin47 analog having substantially the same immunogenic properties as the natural Hin47 protein as provided herein;

(b) means for contacting the analog with the sample to produce a complex comprising the analog and any such antibodies present in the sample; and

(c) means for determining product of the complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction maps of plasmids JB-1031-1-14 and JB-1068-2-2 and the construction of the plasmids for sequence analysis;

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H show the full nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of Hin47 from H. influenzae strain SB33 as well as a partial nucleotide sequence (SEQ ID NO: 3) and a partial deduced amino acid sequence (SEQ ID NO: 4) thereof, the latter being specifically copied by an inventor herein from materials presented in the ASM conference as described above;

FIGS. 3A and 3B show a comparison of the amino acid sequences of H. influenzae Hin47 (SEQ ID NO:2), E. coli htrA (SEQ ID NO: 5), and Salmonella typhimurium htrA (SEQ ID NO:6);

FIGS. 4A, 4B, 4C, 4D and 4E show an alignment of amino acid residues 57 to 256 of Hin47 with certain known proteases (SEQ ID NOS: 7 to 16). Codes are as follows: TON, rat tonin; PKAAB, kallikrein; PTN, trypsin; CHAA, chymotrypsin; EST, elastase: RP2A, rat mast cell protease; SGT, Streptomyces griseus trypsin; SGBE, S. griseus proteinase A; SGA, S.griseus proteinase B; ALP, L.enzymogenes alpha-lytic protease; hin47, res. 57-256 of Hin47. Asterisks(*) denote structurally conserved regions. The catalytic triad residues are indicated by a hash mark (#). `con` refers to regions of structural concensus, among the mammalian proteases;

FIGS. 5A and 5B show the restriction maps for plasmids DS-1011-1-1 and DS-1048-2 which express a Hin47 analog from E.coli and a construction scheme for plasmid DS-1011-1-1 (plasmid pT7/Hin47*);

FIG. 6, comprising panels A and B, shows a process for purifying the Hin47 analog from E. coli according to one embodiment of the present invention (Panel A) and gel analysis (Panel B) of the purified product;

FIG. 7, comprising Panels A, B, and C, shows the protease activities of natural Hin47 and Hin47 analog towards β-casein;

FIG. 8, comprising Panels A, B, and C, shows the stability of natural Hin47 and the Hin47 analog at different temperatures;

FIG. 9, comprising Panels A, B, and C, shows the enzymatic degradation of an H. influenzae recombinant protein by natural Hin47 and the Hin47 analog; and

FIG. 10, comprising Panels A and B, shows the comparative immunogenicity of natural Hin47 and the Hin47 analog in mice;

FIG. 11 shows the amino acid comparison of Hin47 protein isolated from H. influenzae strains SB33 and SB12; and

FIG. 12 shows the purification of the Hin47 analogue H91A from E. coli.

GENERAL DESCRIPTION OF INVENTION

Any Haemophilus strains that have Hin47 genes may be conveniently used to provide the purified and isolated nucleic acid molecules (which may be in the form of DNA molecules), comprising at least a portion coding for Hin47 as typified by embodiments of the present invention. Such strains are generally available from clinical sources and from bacterial culture collections, such as the American Type Culture collection. Such strains include H. influenzae strains and other bacteria that produce a protein capable of generating antibodies that specifically recognize Hin47 fragment or analog thereof. Appropriate strains of Haemophilus may include:

H. influenzae type b strain MinnA;

H. influenzae type b strain Eagan;

H. influenzae non-typable strain SB33;

H. influenzae non-typable strain SB12; or

H. influenzae non-typable strain PAK 12085.

Referring to FIG. 1, there is illustrated restrictions maps of plasmids JB-1031-1-14 and JB-1068-2-2 that contain a portion encoding Hin47 protein from non-typable H. influenzae SB33. The nucleotide sequence of the Hin47 gene was determined and is shown in FIG. 2 along with the deduced amino acid sequence of the Hin47 protein. Referring to FIG. 3, there is shown an amino acid sequence alignment of H. influenzae Hin47 and the serine proteases htrA from Escherichia coli and htrA from Salmonella typhimurium. This alignment for the first time reveals the unexpected discovery of the present applicants that Hin47 is related to bacterial serine proteases and that Hin47 has protease activity. Hin47 has previously been reported to be an adhesin. The discovered protease activity thereof greatly limits the usefulness of natural Hin47 as an immunogen for vaccination and as an antigen in diagnostic uses. The sequence alignment shown in FIG. 3 revealed that the htrA proteins and Hin47 contain a GNSGGAL (SEQ ID NO: 17) sequence between residues 195 and 201 of the mature protein. The consensus sequence of the active site of serine proteases is GDSGGPK (SEQ ID NO: 18) (Brenner, 1988) and the active residue is serine. Thus, Serine-197 in Hin47 was mutated to produce an analog of Hin47 reduced in protease activity, in accordance with one embodiment of the invention. In a particular embodiment, Serine-197 was replaced by alanine. Amino acid residues 57 to 256 of Hin47 were further aligned with known proteases and the active size residues identified from the local homologies surrounding the residues of the catalytic triad (FIG. 4). There is a standard numbering system for serine proteases in which the catalytic triad residues are numbered as His-57, Asp-102 and Ser-195. These correspond to residues His-91, Asp-121 and Ser-197 in the sequential numbering system. Thus, referring to FIG. 4, there is shown a structure-based alignment of ten structurally determined serine proteases (SEQ ID NOS: 7 to 16) in which homologous residues are aligned primarily on the basis of similar locations in three-dimensional space. The location of many of the residues in the hydrophobic core of Hin47, as well as residues around the active site can be aligned reasonably well to identify functional amino acids of the Hin47 protease. Thus, other amino acid residues in Hin47 that contribute to protease activity of the protein include His-91 and Asp-121. In particular embodiments, His-91 may be replaced by alanine, lysine or arginine. In an additional embodiment, Asp-121 may be replaced by alanine or glutamic acid. In an additional embodiment, Serine-197 may be replaced by alanine, serine or threonine. Although the provision of an analog of Hin47 having reduced protease activity has been exemplified herein by particular amino acid substitution within Hin47 protein, the discovery of the protease activity and the methods of Hin47 expression, purification and analysis provided herein, allow for the production of other analogs having at least one other amino acid deleted or replaced or having at least one additional amino acid inserted into the Hin47 protein. In particular applications and embodiments, it may be desirable to simultaneously alter several amino acids of the Hin47 protein to particularly reduce the protease activity of Hin47. The multiple amino acids may be His-91 and Ser-197 and may be deleted or replaced by alanine. In an alternative embodiment, the multiple amino acids may be His-91, Asp-121 and Ser-197 and may be deleted or replaced by alanine. Accordingly, the present invention provides analogs of Hin47 protein having decreased protease activity due to single or multiple amino acid deletions, replacements or additions within the Hin47 protein.

As discussed above, Hin47 shows homology with E. coli htrA or S. tryphimurium htrA, both of which are stress response proteins with serine protease activity. E. coli htrA is inducible by growth at 43.5° C. (ref. 13). We have shown that the E. coli htrA protein is also inducible by growth in 6% ethanol. Hin47 can also be induced by 6% ehtanol and to a lesser extent by temperature reduction at 43.5° C. as described in detail below. This analysis of the expression of Hin47 provides further evidence of the relatedness between this protein and LtrA.

The hin47 gene was also cloned from the non-typable H. influenzae strain SB12 by PCT amplification. Referring to FIG. 11, there is shown an amino acid compariosn between the Hin47 proteins of H. influenzae strains SB12 and SB33. This shows the proteins to be almost identical in amino acid sequence.

Referring to FIG. 5, there is illustrated plasmids DS-1011-1-1 and DS-1048-2 which express a Hin47 analog serine-197→alanine in E. coli. FIG. 6 shows a flow diagram of a method for the purification of the Hin47 analog from E. coli inclusion bodies.

FIG. 7 shows the reduced protease activity of the Hin47 serine-197→alanine analog on the substrate β-casein and demonstrates the analog to have less than about 10% of the protease activity of natural Hin47 protein. Thus, in one embodiment of the invention, there is provided an analog of Hin47 having a protease activity of less than about 10% of the protease activity of natural Hin47 and a such analog may specifically have amino acid Serine-197 replaced by alanine.

Referring to FIG. 8, there is illustrated an analysis of the increased stability of an analog of Hin47 as provided herein. Thus, in one embodiment of the present invention, there is provided an analog of Hin47 protein having increased thermal stability, and such analog may specifically have amino acid serine-197 replaced by alanine.

Referring to FIG. 9, there is illustrated the proteolytic degradation of a non-Hin47 Haemophilus antigen by Hin47 and a Hin47 analog as provided herein. Thus, in accordance with a further embodiment of the present invention, there is provided an analog of Hin47 compatible with a second non-Hin47 protein and such analog may specifically have amino acid Serine-197 replaced by alanine.

Referring to FIG. 10 and Table 1, there is illustrated the comparative immunogenicity of unmodified Hin47 and a Hin47 analog having reduced protease activity in mice. The Hin47 protein and Hin47 analogs S197A and H91A had comparable immunogenicity. Thus, in a particular embodiment, there is provided an analog of Hin47 having reduced protease activity and having substantially the same immunogenic properties of natural Hin47 protein. Such analog may specifically have amino acid Serine-197 replaced by alanine.

Referring to Tables 2 and 3, there is shown the immunoprotective properties of analogs of Hin47 having reduced protease activity against Hib in the infant rat model of bacteraemia and in the active immunization chinchilla model of otitis media according to particular embodiments of the invention, such analog may specifically have amino acids His-91 deleted or replaced by alanine, lysine or arsinine; Asp-121 deleted or replaced by alanine or glutamic acid; Serine-197 replaced by alanine, cysteine or threonine; or combination thereof.

In accordance with another aspect of the present invention, there is provided a vaccine against Haemophilus or other bacterial pathogens that produce Hin47 or a protein capable of inducing antibodies that specifically recognize Hin47, comprising an immunogenically-effective amount of an immunoprotective analog of Hin47 as provided herein or a nucleic acid molecule having a sequence encoding a Hin47 analog as provided herein, and a physiologically-acceptable carrier therefor. The provided analogs also may be used as a carrier protein for hapten, polysaccharides or peptides to make a conjugate vaccine against antigenic determinants unrelated to Hin47.

As will be apparent from the following disclosure, the present invention further provides plasmids and novel strains of bacteria for production of Hin47 analogs as provided herein.

The purified and isolated DNA molecules comprising at least a portion coding for an analog of Haemophilus influenzae Hin47 protein having reduced protease activity compared to natural Hin47 typified by the embodiments described herein, are advantageous as nucleic acid probes for the specific identification of Haemophilus strains in vitro or in vivo. The Hin47 analogs encoded by the DNA molecules provided herein are useful as diagnostic reagents as antigens or for the generation of anti-Hin47 antibodies, antigens for the vaccination against the diseases caused by species of Haemophilus and other bacterial pathogens that produce a protein capable of producing antibodies that specifically recognise Hin47 and for detecting infection by Haemophilus and other such bacteria.

In additional embodiments of the present invention, the Hin47 analogs having reduced protease activity as provided herein may be used as carrier molecules to prepare chimeric molecules and conjugate vaccines (including glycoconjugates) against pathogenic bacteria, including encapsulated bacteria. Thus, for example, glycoconjugates of the present inventions may be applied to vaccinations to confer protection against disease and infection caused by any bacteria having polysaccharide antigens, including lipooligosaccharides (LOS) and PRP. Bacterial pathogens may include, for example, Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis, Salmonella typhi, Streptococcus mutans, Cryptococcus neoformans, Klebsiella, Staphylococcus aureus and Pseudomonas seruginosa. Particular antigens which can be conjugated to analogs of Hin47 and methods to achieve such conjugations are described in applicants published PCT application WO 94/12641 which is hereby incorporated by reference thereto.

In another embodiment, the carrier function of Hin47 analogs may be used, for example, to induce immunity toward abnormal polysaccharides of tumor cells, or to produce anti-tumor antibodies that can be conjugated to chemotherapeutic or bioactive agents.

Accordingly, the present invention provides the primary sequence and the preparation of analogs of Hin47 of H. influenzae that can be used in the prevention and diagnosis of diseases caused by H. influenzae. In particular, the inventors discovered that the Hin47 analogs can elicit protective immune responses against live H. influenzae type b bacterial challenge. Thus, the present inventions have utility in vaccines. The invention also discloses the nucleotide sequences of the genes encoding the Hin47 analogs. These DNA segments may be used to provide an immunogen essentially free from other H. influenzae antigens, such as PRP and lipooligosaccharides (LOS), through the application of recombinant DNA technology. The Hin47 analog protein, may be produced in a suitable expression system, such as E. coli, Haemophilus, Bacillus, Bordetella Fungi, Yeast, Baculovirus, Poxvirus, vaccinia or mammalian expression systems. The present disclosure further provides novel techniques which can be employed for preparing essentially pure Hin47 analogs.

It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, treatment of, for example, Haemophilus infections, and infections with other bacterial pathogens that produce proteins capable of producing antibodies that specifically recognize Hin47 and the generation of immunological reagents. A further non-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may be prepared from Hin47 analogs as disclosed herein. The vaccine elicits an immune response in a subject which produces antibodies, including anti-Hin47 antibodies and antibodies that are opsonizing or bactericidal. Should the vaccinated subject be challenged by Haemophilus or other bacteria that produce proteins capable of producing antibodies that specifically recognize Hin47, the antibodies bind to and inactive the bacterium. Furthermore, opsonizing or bactericidal anti-Hin47 antibodies may also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions or emulsions. The Hin47 analogs may be mixed with pharmaceutically acceptable excipients which are compatible with the Hin47 analog. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof. Methods of achieving adjuvant effect include the use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline. Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the, immunogenic composition may be administered to mucosal surfaces by, for example, the nasal, or oral (intragastric) routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the Hin47 analogs. The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the Hin47 analogs. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of the host.

The concentration of antigen in an immunogenic composition according to the invention is in general about 1 to 95%. A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.

The nucleic acid molecules encoding the Hin47 analog of the present invention may also be used directly for immunization by administration of the DNA directly, for example, by injection for genetic immunization or by constructing a live vector, such as Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus. A discussion of some live vectors that have been used to carry heterologous antigens to the immune system are discussed in, for example, O'Hagan (1992). Processes for the direct injection of DNA into test subjects for genetic immunization are described in, for example, Ulmer et al, 1993.

2. Immunoassays

The Hin47 analogs of the present invention are useful as immunogens for the generation of anti-Hin47 antibodies, as antigens in immunoassays including enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of anti-bacterial, Haemophilus, and anti-Hin47 antibodies. In ELISA assays, the Hin47 analogs, are immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed Hin47 analogs, a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conductive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from 2 to 4 hours, at temperatures such as of the order of about 25° to 37° C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or borate buffer. Following formation of specific immunocomplexes between the test sample and the bound Hin47 analogs, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a colour development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of colour generation using, for example, a visible spectra spectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleic acid molecules of the present invention, having the sequence of the hin47 analog gene, allow for the identification and cloning of the Hin47 genes from any species of Haemophilus and other bacteria that produce proteins capable of producing antibodies that specifically recognize Hin47.

The nucleic acid molecules having the sequence encoding the Hin47 analog of the present invention are useful for their ability to selectively form duplex molecules with complementary stretches of other hin47 genes. Depending on the application, a variety of hybridization conditions may be employed to achieve varying degrees of selectively of the probe toward the other hin47 genes. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes, such as low salt and/or high temperature conditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures of between about 50° to 70° C. For some applications, less stringent hybridization conditions are required, such as 0.15 M to 0.9 M salt, at temperatures ranging from between about 20° C. to 55° C. Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be a method of choice depending on the desired results.

In a clinical diagnostic embodiment, the nucleic acid molecules encoding the hin47 genes of the present invention may be used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of providing a detectable signal. In some diagnostic embodiments, an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of a radioactive tag may be used. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing hin47 gene sequences.

The nucleic acid molecules comprising hin47 genes of the present invention are useful as hybridization probes in solution hybridizations and in embodiments employing solid-phase procedures. In embodiments involving solid-phase procedures, the test DNA (or RNA) from samples, such as clinical samples, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. The fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid sequences of the hin47 genes of the present invention under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required depending on, for example, the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe etc. Following washing of the hybridization surface so as to remove non-specifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

4. Expression of the Genes encoding analogs of Hin47 having reduced protease activity

Vectors perhaps containing replicon and control sequences which are derived from species compatible with the host cell may be used for the expression of the Hin47 analog genes as provided herein in expression systems. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be transformed using pBR322 which contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the host cell for expression of its own proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host can be used as a transforming vector in connection with these hosts. For example, the phage in lambda GEM™-11 may be utilized in making recombinant phage vectors which can be used to transform host cells, such as E. coli LE392.

Promoters commonly used in recombinant DNA construction include the β-lactamase penicillinase) and lactose promoter systems (Chang et al, 1979; Goeddel et al, 1980) and other microbial promoters, such as the T7 promoter system (U.S. Pat. No. 4,952,496). Details concerning the nucleotide sequences of promoters are known, enabling a skilled worker to ligate them functionally with plasmid vectors. The particular promoter used will generally be a matter of choice depending upon the desired results. Hosts that are appropriate for expression of the Hin47 analogs include E. coli, Bacillus species, Haemophilus bordetella fungi, yeast, mammalian cells or the vaculovirus expression system may be used.

Thus, in accordance with the invention, it may be preferred to make the Hin47 analog protein by recombinant methods. Particularly desirable hosts for expression in this regard include Gram positive bacteria which do not have LPS and are therefore endotoxin free. Such hosts include species of Bacillus and may be particularly useful for the production of non-pyrogenic Hin47 analog.

Biological Deposits

Plasmid DS-1011-1-1 (pT7/Hin47*) that contains a portion coding for a Hin47 analog that is described and referred to herein has been deposited with the American Type Culture Collection (ATCC) located at Rockville, Md., USA, pursuant to the Budapest Treaty and prior to the filing of this continuation-in-part application on Jul. 27, 1994 under Accession No. 75845. Samples of the deposited plasmid will become available to the public upon grant of a patent based upon this United States patent application. The invention described and claimed herein is not to be limited in scope by plasmid deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as described in this application are within the scope of the invention.

EXAMPLES

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the ability of those skilled in the art.

Example 1

This Example illustrates the cloning of the hin47 gene from non-typable H. influenzae strain SB33.

Chromosomal DNA was prepared from H. influenzae strain SB33, and an EMBL3 library was prepared and screened with a labelled oligonucleotide probe specific for the 5'-end of hin47. Non-typable H. influenzae strain SB33 was grown on Mueller-Hinton agar or in brain heart infusion broth as described by Harkness et al, 1992. Chromosomal DNA was prepared as follows: cells from 50 ml of culture were pelleted by centrifugation at 5000 rpm for 15 to 20 min, at 4° C., in a Sorvall RC-3B centrifuge. The cell pellet was resuspended in 10 ml of TE (10 mM Tris/HCl, 1 mM EDTA, pH 7.5), pronase was added to 500 μg ml⁻¹ and SDS to 1%. The sample was incubated at 37° C. until a clear lysate was obtained. The lysate was gently extracted once with Tris-saturated phenol (pH 7.4), once with Tris-saturated phenol/chloroform (1:1) and once with chloroform. The final aqueous phase was dialysed at 4° C. for 24 h against 1M NaCl, followed by 24 h against TE.

An EMBL3 library was prepared by partial digestion of SB33 chromosomal DNA with Sau3A I, followed by size fractionation either on a 10 to 30% sucrose gradient in TNE (20 mM Tris/HCl, 5 mM NaCl, 1 mM EDTA, pH 8.0) or by preparative gel electrophoresis. Fractions containing DNA fragments greater than 5 kb in length were pooled, precipitated and ligated with BamH I arms of EMBL3 (Promega). The ligation mixture was packaged using a Gigapack II packaging kit and plated onto E. coli LE392 cells. The libraries were amplified and stored at 4° C. in the presence of 0.3% chloroform.

Plaques were lifted on nitrocellulose filters for hybridization with a ³² P-labelled oligonucleotide probe (3026.SL). The oligonucleotide sequence was ATGAAAAAAACACGTTTTGTATTAAATAGTATTGCACTTGG (SEQ ID NO: 3) corresponding to the N-terminal amino acid sequence MKKTRFVLNSIALG (SEQ ID NO: 19). Phage DNA was prepared from putative plaques and the insert DNA was excised by Sal I digestion and cloned into pUC8-BgXb digested with Sal I. Plasmids JB-1031-1-14 and JB-1068-2-2 (FIG. 1) were selected for further analysis.

Example 2

This Example illustrates the characterization and sequence analysis of the hin47 gene and deduced amino acid sequence of the Hin47 protein from NTHi strain SB33.

Restriction mapping and Southern blot analysis of clones JB-1031-1-14 and JB-1068-2-2 localized the hin47 gene on a 4.7 kb BamH I/BamH I or a 2.7 kb Bam H I/Pst I DNA fragment. The 4.7 kb BamH I/BamH I fragment from JB-1068-2-2 was subcloned into pUC8/BgXb generating plasmid DS-755-1. The 3.1 kb Bam H I to Xba I fragment of DS-755-1 was subcloned into pUC18 generating plasmid JB-1165-1 which has restriction sites suitable for the Erase-a-base (Promega) procedure (FIG. 1). This technique generates successive clones with increasing truncations of insert DNA, with the deletions occurring from the same end. The resultant nested set of clones can be sequenced rapidly using a universal primer.

DNA from plasmid JB-1165-1 was digested with BamH I and Sac I and subjected to exoIII digestion using an Erase-a-base kit. The resultant set of truncated plasmids was analysed by agarose gel electrophoresis and representative plasmids were selected for sequence analysis.

Plasmid DNA for sequencing was prepared by a modification of the procedure of Holmes and Quigley, 1981. Briefly, the cell pellet from 50 ml of culture was resuspended in 10 ml STET (8% sucrose, 5% Triton X-100, 50 mM EDTA, and 50 mM Tris/HCl, pH 8.0), lysozyme (2.5 mg) was added and the mixture was boiled for 2 min. The sample was spun at 14,000 rpm in a Sorvall RC 5B for 20 minutes and the supernatant was precipitated with an equal volume of isopropanol, washed with 70% ethanol then absolute ethanol, and then air dried. The pellet was resuspended in 0.9 ml of TE, then 20 μl of 5 mg ml⁻¹ RNAse A were added, and the mixture was incubated at 37° C. for 15 min. After the addition of 500 μl of 1.5M NaCl/30% PEG, the mixture was incubated on ice for 30 min and the DNA was pelleted by centrifugation in an Eppendorf microfuge for 10 min. The pellet was resuspended in 400 μl of TE and extracted twice with Tris-saturated phenol (pH 7.4), twice with Tris-saturated phenol/chloroform (1:1) and twice with chloroform. The DNA was precipitated by adding 40 μl of 3M ammonium acetate and 1 ml of ethanol, washed with 70% ethanol and resuspended in distilled water.

DNA samples were sequenced using the ABI model 370A DNA sequencer and the dye terminator chemistry. The universal reverse primer was used with the nested set of clones to determine the sequence of the hin47 coding strand. Oligonucleotide primers of approximately 25 bases in length were used to confirm the sequence of the non-coding strand. The nucleotide sequence of the SB33 hin47 gene and the deduced amino acid sequence of the Hin47 protein are shown in FIG. 2. The nucleotide and N-terminal amino acid sequences of Hin47 presented at the ASM meeting, New Orleans, May 26 to 30, 1992 are indicated in lower case on FIG. 2. The amino terminal sequences of the SB33 Hin47 and this presented sequence are identical, establishing the identity of the cloned gene as hin47.

Example 3

This Example describes the discovery of serine protease activity of Hin47 protein.

The deduced amino acid sequence of Hin47 protein determined in Example 2 above was compared with all other known proteins in the Genbank data base. As described above, Hin47 protein is described in published PCT applications WO 94/00149, WO 92/11367 and WO 92/10936 to be an adhesion molecule of Haemophilus. It was, therefore, a surprising and unexpected discovery of the present invention that Hin47 protein has significant amino acid homology (55%) with the serine proteases E. coli htrA and S. typhimurium htrA and other proteases. These amino acid sequence homologies are shown in FIGS. 3 and 4. Furthermore, Hin47 protein was found to autodigest unless it was stored in the presence of a serine protease inhibitor, such as Pefablock.

Example 4

This Example illustrates the generation of the mutant hin47 gene by site-directed mutagenesis.

As explained above, H. influenzae Hin 47, E. coli htrA, and S. typimurium htrA are all serine proteases. The consensus sequence of the active site of serine proteases is GDSGGPK (SEQ ID NO: 18) [Brenner, 1988] with serine being the active residue. The htrA proteins both have a GNSGGAL (SEQ ID NO: 17) sequence and in H. influenzae Hin47, there is the identical sequence between residues 195 and 201 of the mature protein. Thus, the serine residue at position 197 was selected for site-directed mutageneis, to produce an analog of Hin47 with reduced protease activity.

An oligonucleotide CGCTCCACCAGCATTACCGCGG (SEQ ID NO: 20) was synthesized which would change the serine residue at 197 to an alanine. The hin47 gene was cloned into M13mp18 generating clone DS-981-3 and mutagenesis was performed using the Amersham In Vitro Site-Directed Mutagenesis kit. Clone DS-991-8 was confirmed by sequence analysis to contain the mutation Serine-197 to Alanine. This mutant hin47 gene is designated hin47*. Using appropriate oligonucleotides, the serine residue at 197 was changed to a cysteine (mutant S197C) and a threonine (mutant S197T)

In addition a comparison of the amino acid sequence of Hin47 with other proteases (as shown in FIG. 4) revealed that amino acids His-91 and Asp-121 are sites appropriate for mutagenesis to produce an analog of Hin47 with reduced protease activity. By mutagenesis methods analogous to those described above, His-91 and/or Asp-121 were deleted or replaced by different amino acids. Such amino acid replacements included His-91 to Alanine (mutant H91A) and Arginine (mutant H91R) and Asp-121 to Alanine (mutant D121A) and Glutamic acid (mutant D121E). Oligonucleotides to effect such mutagenesis included:

His-91→Ala-91 5' ATCAATAACAGCATTATTGGT 3' (SEQ ID NO: 21)

Asp-121→Ala-121 5' TAATGCAATTGCTGATAGTTC3' (SEQ ID NO: 22).

Corresponding oligonucleotides were employed to effect the other mutations. Multiple mutations also were effected in which His-91 and serine-197 both were replaced by Alanine (mutant H91A/S197A) and His-91, Alp-121 and Ser-197 were all replaced by Alanine (mutant H91A/D121A/S197A).

These additional mutants were produced, extracted, purified and tested for protease activity as described for the Hin47* material in the succeeding examples.

Many serine proteases are secreted in an inactive (`zymogen`) form, and require clipping to expose their active sites. N terminal sequence analysis of mature natural Hin47 protein suggested the cleavage of the preprotein to occur at KFFFG DRFAEQ (SEQ ID NO: 23). Modifications of amino acids that prevent cleavage of the molecule to produce the active protease molecule can produce an analog of Hin47 having reduced protease activity.

Example 5

This Example illustrates the construction of plasmids expressing Hin47 Ser-197→alanine analog from E. coli.

The mutated hin47* gene from plasmid DS-991-8 was cloned into the pT7-7 expression vector to generate plasmid DS-1011-1-1 (FIG. 5). E. coli strain BL21/DE3 was transformed to generate E. coli strain DS-1018-3-1 which expresses Hin47 Ser-197→alanine analog upon induction.

In order to utilize tetracycline selection, the hin47* gene was cloned into pBR328. The Bgl II/Cla I T7/hin47* gene fragment from DS-1011-1-1 was cloned into pEVvrf1 (Young and Davis, 1985) in order to generate a Bgl II/BamH I fragment which could be cloned into pUC-4K (Pharmacia) digested with BamH I. The resultant clone DS-1034-3 was digested with EcoR I and the T7/hin47* gene fragment cloned into pBR328 (Boehringer Mannheim Corporation) to generate plasmids DS-1048-2 and DS-1067-2. Electroporation of plasmid DNA into E. coli strain BL21/DE3 resulted in strains DS-1071-1-1 and DS-1071-3-1 which express the Hin47 Ser-197→alanine analog.

Example 6

This Example illustrates the expression of Hin47 Ser-197→alanine analog from E. coli.

An overnight culture of strains DS-1018-3-1, DS-1071-1-1, or DS-1071-3-1 grown overnight in NZCYM media+3% dextrose+antibiotics (ampicillin at 25 μg ml⁻¹ or tetracycline at 10 μg ml⁻¹), at 37° C., with shaking. A 1:40 dilution of the overnight culture was inoculated into the same medium and grown at 37° C. with shaking until the absorbance was A₅₇₈ approximately 0.3. A 1/10 volume of 10% lactose was then added to induce expression from the T7 promoter. Cell samples were harvested about 4 hours after induction by centrifuging culture samples at 5000 rpm for 10 min in a Sorvall RC-3B, at 4° C.

Example 7

This Example illustrates the extraction and purification of Hin47.

Hin47 was expressed as soluble protein in E. coli. The cell pellet from a 250 ml culture, prepared as described in Example 6, was resuspended in 40 ml of 50 mM Tris-HCl, pH 8.0, and disrupted by sonication (3×10 min, 70% duty circle). The extract was centrifuged at 20,000×g and the resulting supernatant which contained >95% of the soluble Hin47 protein was retained. This fraction was called "Hin47-extract".

This Hin47-extract was further purified on a DEAE Sephacel column. Forty ml of the Hin47-extract was applied onto a 20-ml DEAE Sephacel column equilibrated in 50 mM Tris-HCl, pH 8.0. Hin47 bound to the column under these conditions. The column was washed with 100 ml of 50 mM Tris-HCl, pH 8.0, and then washed with 100 ml of 50 mM Tris-HCl, pH 8.0 containing 20 mM NaCl. Hin47 was then eluted with 50 mM Tris-HCl, pH 8.0, containing 40 mM NaCl. The amount of the Hin47 in the fractions was determined by the BCA protein assay. The purity of Hin47 was assessed by SDS-PAGE analysis. The fractions containing Hin47 were combined and stored at -20° C.

Only the H91A mutant was as soluble as the wild-type Hin47 protein, most of the other mutants being produced as inclusion bodies.

Example 8

This Example illustrates the extraction and purification of Hin47 Ser-197→alanine analog.

Hin47 Ser-197→alanine analog was expressed in inclusion bodies in E. coli. The cell pellet from a 250 ml culture, prepared as described in Example 6, was resuspended in 40 ml of 50 mM Tris-HCl, pH 8.0, and disrupted by sonication (3×10 min, 70% duty circle). The extract was centrifuged at 20,000×g and the resulting pellet was saved. The pellet was re-extracted with 40 ml of 50 mM Tris-HCl, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was sonicated 10 min at 70% duty circle. The extract was centrifuged at 300×g for 5 min. The resultant supernatant was centrifuged again at 20,000×g for 30 min and the resultant pellet was saved. The pellet was resuspended in 50 mM Tris-HCl, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was then mixed with 50 mM Tris-HCl, pH 8.0 containing 8 M urea. The final urea concentration in the mixture was adjusted to 2 M with 50 mM Tris-HCl, pH 8.0. Hin47 Ser-197→alanine analog was completely solubilized under these conditions. The final volume of the solution was 20 ml. This fraction is called "Hin47 analog-extract". The Hin47 analog-extract was further purified on a DEAE Sephacel column. Twenty ml of Hin47 analog-extract was applied onto a 10 ml DEAE Sephacel column equilibrated in 50 mM Tris-HCl, pH 8.0. Hin47 Ser-197→alanine analog bound to the column under there conditions. The column was washed with 50 mM Tris-HCl, pH 8.0, and Hin47 analog was eluted with 50 mM Tris-HCl, pH 8.0, and containing 30 mM NaCl. The amount of Hin47 analog in the fractions was determined by the BCA protein assay. The purity of Hin47 analog was assessed by SDS-PAGE analysis (FIG. 6). The fractions containing Hin47 analog were combined and stored at -20° C.

Example 9

This Example illustrates the protease activity of Hin47 and Hin47 Ser-197→alanine analog.

The enzymatic activity of Hin47 and Hin47 Ser-197→alanine analog was analyzed using β-casein as a substrate (FIG. 7). The reaction mixtures contained 5 μg of β-casein and either Hin47 or Hin47 analog. The reaction was carried out at 37° C. for two hours, and then stopped by adding the SDS-sample buffer and instantly heating the sample at 100° C. for 5 min. The aliquots were analyzed by SDS-PAGE. As shown in FIG. 7, digestion of β-casein by Hin47 was more obvious after two hours (panel A, lane 1) in comparison to the fractions containing Hin47 analog (panel A, lane 2) or without any exogenous proteins (panel A, lane 3). The presence of Hin47 or Hin47 analog in these mixtures were confirmed by immuno-blotting using a monoclonal antibody to Hin47 (FIG. 7, panel C, lanes 1 and 2).

The protease activities of Hin47 and Hin47 Ser-197→alanine analog were also compared by analyzing the autodigestion of Hin47 or Hin47 analog at 4° C. and -20° C. The purified Hin47 or analog were stored at either 4° C. or -20° C. for up to 20 days. Aliquots were taken on days 0, 10 and 20 and the stability of Hin47 or analog was analyzed by immuno-blotting using a Hin47 monoclonal antibody (FIG. 8). The analog was much more stable than Hin47 up to 20 days when stored at either 4° C. or -20° C.

To further examine the protease activity of the Hin47 Ser-197→alanine analog, the ability of Hin47 or analog to degrade an 80-kDa H. influenzae recombinant antigen was examined. Thus, a mixed antigen study was performed to determine the proteolytic effect of Hin47 or Hin47 analog on another antigen. An 80 kDa H. influenzae recombinant protein (TBP1) was chosen for this study in order to distinguish it from the Hin47 or analog protein (47 kDa). Five minutes were formulated as follows: 80-kDa protein alone; 80-kDa protein+Hin47; 80-kDa protein+analog; Hin47 alone; and analog alone. The amount of each protein in the mixture was 5 μg. The mixtures were stored at 4° C. up to four weeks. Aliquots were taken on days 0, 7, 14 and 28 for analysis by SDS-PAGE (FIG. 9). Both the 80 kDa protein and Hin47 were largely degraded after one week (lane 2 and 4). In contrast, the 80 kDa protein in combination with Hin47 analog remained intact after one week, and showed only slight degradation even after four weeks (lane 3).

The residual protease activity of other Hin47 analogues was assessed using the digestion of β-casein as described by Lipinska et al (ref. 13) and the results of which are shown in Table 3. Only one mutant (D121E) was found to retain serine protease activity.

Example 10

This Example illustrates the comparative immunogenicity of Hin47 and Hin47 analog in mice.

The results of a study to determine the comparative immunogenicity of Hin47 and the Hin47 Ser-197→alanine analog are shown in FIG. 10. Thus, groups of five Balb/c mice were injected three times (as indicated by arrows) s.c. on days 1, 29 and 43 with 1-μg dose of either Hin47 or Hin47 analog in the presence of AlPO₄ (1.5 mg per dose). Blood samples were taken on days 14, 28, 42 and 56 (as indicated by bleeds 1, 2, 3 and 4, respectively) for analyzing the anti-Hin47 antibody titers by EIAs. The determination of anti-Hin47 antibodies in mouse sera was performed as described by Panezutti et al. (1993). Microtiter wells were coated with 1 μg of either Hin47 or Hin47 analog for 16 hours at room temperature. The plates were then blocked with 0.1% (w/v) bovine serum albumin in PBS. The mouse sera were serially diluted, added to the wells, then incubated for one hour at room temperature. Affinity-purified F(ab')₂ fragments of goat anti-mouse IgG (Fc specific) antibody conjugated to horseradish peroxidase were used as the second antibody. The reactions were developed using tetramethylbenzidine (TMB/H₂ O₂) and absorbencies were measured at 450 nm (using 540 nm as a reference wavelength) in a Flow Multiskan MCC microplate reader. The reactive titer of an antiserum was defined as the reciprocal of the dilution consistently showing a two-fold increase in absorbance over that obtained with the pre-bleed serum sample. As can be seen from FIG. 10, both Hin47 and the Hin47 analog elicited comparable IgG titers in mice regardless of whether Hin47 or mutant was used as an antigen in EIAs.

Immunogenicity studies were also performed using the H91A Hin47 analogue. This analogue was found to produce an immune response equivalent to that of the S197A Hin47 analogue.

To further examine the immune response to Hin47 or the Hin47 Ser-197→analine analog, the subclasses of anti-Hin47 IgG in mouse sera were determined. Microtiter wells were coated with 1 μg of purified Hin47 or analog. The final bleed of mouse serum samples from the comparative immunogenicity study (as described above) were pooled and tested in EIAs. Rat anti-mouse IgG₁, IgG_(2a), IgG_(2b) antibodies conjugated horseradish peroxidase and rabbit anti-mouse IgG₃ conjugated to horseradish peroxidase were used as reagents in EIAs. The working dilution of each conjugate was determined using purified antibody subclasses to avoid cross reactivity. The reactive titers were determined as described above. As shown in Table 1 below, the IgG-subclass profile induced in mice by either Hin47 or Hin47 analog were identical, regardless of whether Hin47 or analog was used as a solid antigen in the EIAs. The predominant IgG response in both groups of mouse sera was of the IgG₁ isotype. Hence, the Hin47 analog exhibited substantially the same immunogenic properties as the natural protein.

Example 11

This Example illustrates the immunoprotective properties of Hin47 and Hin47 Ser-197→alanine analog.

The immunoprotective properties of Hin47 and the Hin47 Ser-197→alanine analog were analyzed by the ability of Hin47 specific antisera to protect infant rats against H. influenzae type b strain MinnA in the bacteremia model. The results of this study are shown in Table 2 below. Groups of nine 6-day old Wistar infant rats were inoculated subcutaneously (s.c.) on the dorsum close to the neck with 0.1 mL of either a rabbit anti-Hin47 analog antiserum or the corresponding prebleed serum. Twenty-four hours later, the animals were challenged intrapertioneally (i.p.) with 700 cfu of freshly grown Hib strain MinnA. Blood samples were collected 20 hours post-challenge and plated onto chocolate agar plates. Bacterial colonies were counted after 24 hours. As shown in Table 2, three out of nine animals in the group inoculated with anti-Hin47 analog antiserum did not show any bacteremia in their blood. Only one mouse in the group inoculated with anti-Hin47 analog antiserum (11%) had a higher bacteria recovery from the blood sample compared to mice inoculated with prebleed serum. In contrast, bacteria were recovered from all the nine mice inoculated with pre-bleed serum. Four out of nine animals (44%) in the group inoculated with pre-bleed serum showed high levels (500 to 1,000) of bacteria recovered in blood samples.

The infant rat model of bacteremia, was used to assess the protection afforded by anti-Hin47 or anti-Hin47 mutant antisera against bacteremia caused by H. influenzae type b infection. 6/10 infant rats were protected by antisera raised against each of wild-type Hin47, H91A Hin47, and S197A Hin47 analogues.

Example 12

This Example illustrates the induction of Hin47 under stress conditions.

H. influenzae strain Eagan was grown at 37° C. to an A₅₉₀≈0.3 in brain heart infusion broth (BHI) containing hemin (2 μg ml⁻ 1) and NAD (2 μg ml⁻ 1). The sample was aliquotted and grown at 37° C., 42° C., 43.5° C., or in the presence of 6% ethanol, 0.2M NaCl, or 0.3M NaCl. E. coli strain JM109 was grown at 37° C. to an A₅₉₀ of ≈0.3 in YT strain JM109 was grown at 37° C. to an A₅₉₀ of ≈0.3 in YT media and aliquotted as described. Samples were collected at 0 min, 20 min, 40 min, 60 min, and 90 min and analyzed by OD and SDS-PGE/Western blot. Guinea pig antisera which recognized both H. influenzae Hin47 and E. coli htrA was used for Western blot analysis. The E. coli htrA protein was produced in large quantities when the organism was grown at 43.5° C. and a small amount of the H influenzae Hin47 protein can be observed. With growth in media containing 6% ethanol, both the E. coli htrA and the H. influenzae Hin47 proteins are induced. The high salt conditions were insufficient to induce either protein. These results indicate that Hin47 is a stress response protein in H. influenzae, inducible under similar conditions to the E. coli htrA protein.

Example 13

This Example illustrates the purification of the H91A Hin47 protein.

The soluble H91A mutant was purified essentially as described for the wild-type Hin47 in Example 7, with the addition of a hydroxylapatite (HAP) column. The HAP column was equilibrated in 10 mM sodium phosphate buffer (pH 8.0) and the run-through from the DEAE column was loaded. The H91A Hin47 bound to the HAP column and contaminating proteins were removed by washing the column with 175 mM sodium phosphate buffer. The H91A Hin47 protein was eluted with 300 mM sodium phosphate buffer (pH 8.0) was stored at -20° C.

Example 14

This Example illustrates the protection studies with Hin47 and Hin47 mutants in the chinchilla model of otitis media.

The chinchilla model of otitis media (ref. 14) was used to assess the protection induced by active immunization with wild-type Hin47, H91A Hin47, or S197A Hin47.

Chinchillas (˜500 g weight) were immunized i.m. three times with 30 μg/dose of Hin47 or Hin47 mutant (H91A or S197A) adjuvanted in AIPO4, on days 1, 28 and 42. The animals were challenged on day 56, through the bulla, with 50-1000 cfu of virulent NTHi strain SB12 organisms. Animals were monitored by tympanometry and otoscopic examination and at 4 days post-challenge, middle ear fluids were aspirated and plated on chocolate agar. Bacterial colonies were counted after 24 h. The wild-type Hin47 and H91A Hin47 proteins afforded protection to ˜50% of the animals, but the S197A Hin47 was non-protective in this model (Table 3).

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides novel analogs of Haemophilus influenzae Hin47 protein which have a decreased protease activity of less than about 10% of that of the natural Hin47 protein as well as isolated and purified DNA molecules encoding the same. Modifications are possible within the scope of this inventions.

                  TABLE 1                                                          ______________________________________                                         Hin47 IgG titers in mouse immune sera                                                   IgG titers in Group #1                                                                         IgG titers in Group #2                                IgG Suclass                                                                             To Hin47  To Mutant To Hin47                                                                               To Mutant                                 ______________________________________                                         IgG (H + L)                                                                             102,400   102,400   102,400 102,400                                     IgG.sub.1 25,600 25,600 25,600 25,600                                          IgG.sub.2a <100 <100 <100 <100                                                 IgG.sub.2b 400 400 400 400                                                     IgG.sub.3 <100 <100 <100 <100                                                ______________________________________                                          Group #1: Immune sera were pooled from a group of five mice received Hin4      immunization.                                                                  Group #2: Immune sera were pooled from a group of five mice received Hin4      mutant immunization.                                                     

Plates were coated with either Hin47 or mutant protein.

                  TABLE 2                                                          ______________________________________                                         Protective ability of rabbit Anti-Hin47 Mutant                                   antiserum against Hib in infant rat model of bacteremia                             Number of Animals                                                         cfu of Bacteria/2.5 μL Blood                                                             Av.50    Av.200  Av.650  Total                                    Antibody Av.0 (10-100) (100-300) (300-1,000) Animals                         ______________________________________                                         Anti-  3       3        2       1       9                                        Hin47*                                                                         Prebleed 0 4 1 4 9                                                           ______________________________________                                    

Groups of nine 6-day old infant rats were immunized s.c. with either a rabbit anti-Hin47 mutant antiserum or the corresponding prebleed serum. Animals were challenged i.p. with 700 cfu H. influenzae strain MinnA after 24 hours. The blood samples were taken at 20 hours after the challenge.

Anti-Hin47* antibody: rabbit immune serum raised against purified Hin47 mutant in CFA/IFA.

Average bacteria recovery from immunized group: 100 cfu per 2.5 μL of blood; from control group: 290 cfu per 2.5 μL of blood.

                  TABLE 3                                                          ______________________________________                                         Characterization of Hin47 mutants                                                                             Protection -                                                                           Protection -                              Mutant Protease.sup.a Solubility.sup.b rat.sup.c chinchilla.sup.d            ______________________________________                                         WILD-TYPE                                                                               +         +         +       ±                                        H91A - + + ±                                                                H91R - - .sup.  ND.sup.e ND                                                    D121A - - ND ND                                                                D121E + - ND ND                                                                S197A - - + -                                                                  S197C - ± ND ND                                                             S197T - ± ND ND                                                             H91A/S197A - - ND ND                                                           H91A/D121A/ - - ND ND                                                          S197A                                                                        ______________________________________                                          .sup.a Protease activity is measured by the ability to digest the              substrate casein.                                                              .sup.b Solubility indicates production as a soluble protein (+) or             inclusion bodies (-).                                                          .sup.c Protection in the infant rat passive model of bacteremia.               .sup.d Protection in the chinchilla model of otitis media.                     .sup.e ND is not determined                                              

REFERENCE LIST

1. Zangwill et al, 1993 MMWR 42:1-15.

2. Schoendorf et al, 1994 Pediatrics 93:663-8.

3. Brenner et al, 1988 Nature 334:528-530.

4. O'Hagan 1992 Clin. Pharmokinet. 22:1-10.

5. Ulmer et al, 1993 Curr. Opinion. Invest. Drugs 2:983-989.

6. Chang et al, 1978 Nature 275:617.

7. Goeddel et al 1980 Nucl. Acid. Res. 8:4057.

8. Harkness et al, 1992 J. Bacteriol. 174:2425-2430.

9. Loeb et al, 1987 Infec. Immun. 55:2612-2618.

10. Holmes and Quigley 1981. Analyt. Biochem. 114:193-197.

11. Young and Davis 1985 Gene 38:31-38.

12. Panezutti et al, 1993 Infec. Immun. 61:1867-72.

13. Lipinska et al, 1985 Bacteriol. 171:1574-1584.

14. Barenkamp et al, 1986 Infect. Immun. 52:572-578.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 23                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2894 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                            - - GGATCCGTTA ATACTGAAAT AAATGGCACA CCTTTTTCAC GCATTTGGGC AA -             #GTACAGCA     60                                                                  - - CTGGTTTTTG CCATTTGCAT TAAAGAGAAT AATGCTTCCT GCATACGAGC AC -             #CACCACTC    120                                                                  - - GCAGAGAAAC ATACAAACGG ACAATTCATT TCCATCGCTT TTTCAGCCGC TT -             #TAACAAAT    180                                                                  - - TTTGCACCAA CTACAGAACC CATTGAACCG CCCATAAAAG CAAAGTTCGA TG -             #CAGCCACA    240                                                                  - - ACAATTGGCA TATCATAAAG TGTACCTGTC ATAGTAATTA GCGCATCTTT CT -             #CGCCCGTT    300                                                                  - - TCTTTTTGTG CCGCATTGAT ACGATCTTTA TATTTCTTTA AATCTTTAAA TT -             #TTAAAATA    360                                                                  - - TCTTTTGGTT CTAAATCTGC CGCAATTTCT TGGCTTGAAT CTTCGTCCAA TA -             #AATTTAAT    420                                                                  - - AAACGCTCAC GAGCATCAAT ACGCATATGA TGACCACATT TCGGGCAAAC AT -             #ACAGATTA    480                                                                  - - CGTTTGAGTT CTTCACTATA AAGTACTTGT TCACAAGCAG TACATTTTGT CC -             #ATACGCCT    540                                                                  - - TCTGGCACAT TGGCTTTTCG AGTGGAAGAA GAAGGACTTT TACTAAAAAT TC -             #GGTTAATC    600                                                                  - - CAGCTCATTT TTTGACCTTT TTATTGACTA GAAAATTGCG CGTATTAGAA CA -             #TAAATTTA    660                                                                  - - TAGAATTTGC TACTTGTAAG ACCGTTTTTG TACTGCTCCG ATTTCCTTTT AA -             #ACAAGATA    720                                                                  - - ATTTGCTCTC CTCTTATTGA ACATTTTTTT TATTTTTTTG TCTTACTGAC CA -             #CGTTATCT    780                                                                  - - GAAATTTATT TTGGAGTATT TATGAAAAAA ACACGTTTTG TACTAAATAG TA -             #TTGCACTT    840                                                                  - - GGATTAAGTG TATTAAGCAC ATCATTTGTT GCTCAAGCCA CTTTGCCAAG TT -             #TTGTTTCG    900                                                                  - - GAACAAAACA GTCTTGCACC AATGTTAGAA AAAGTACAAC CTGCCGTTGT CA -             #CTCTTTCC    960                                                                  - - GTTGAAGGAA AAGCTAAAGT AGATTCTCGT TCTCCTTTCC TAGACGATAT TC -             #CTGAAGAA   1020                                                                  - - TTTAAATTCT TCTTTGGCGA TCGTTTTGCC GAACAATTTG GTGGACGTGG AG -             #AATCAAAG   1080                                                                  - - CGTAACTTCC GTGGTTTAGG TTCTGGTGTC ATTATTAATG CAAGCAAAGG CT -             #ATGTTTTA   1140                                                                  - - ACCAATAATC ATGTTATTGA TGAAGCTGAT AAAATTACCG TGCAATTACA AG -             #ATGGGCGT   1200                                                                  - - GAATTTAAAG CAAAATTAGT GGGTAAAGAT GAACTATCAG ATATTGCATT AG -             #TACAGCTT   1260                                                                  - - GAAAAACCAA GTAATTTAAC AGAAATCAAA TTTGCTGATT CCGACAAATT AC -             #GCGTAGGC   1320                                                                  - - GATTTCACTG TTGCAATCGG TAATCCATTT GGTTTAGGTC AAACTGTGAC AT -             #CAGGTATT   1380                                                                  - - GTTTCTGCAT TGGGTCGTTC AACAGGTTCT GACAGTGGCA CTTATGAAAA CT -             #ATATTCAA   1440                                                                  - - ACCGATGCAG CAGTAAACCG CGGTAATTCG GGTGGAGCGT TAGTAAACTT AA -             #ATGGCGAA   1500                                                                  - - CTTATTGGAA TTAATACCGC AATTATTTCT CCAAGCGGTG GCAATGCAGG AA -             #TTGCCTTT   1560                                                                  - - GCGATTCCAA GTAATCAAGC AAGCAATTTA GTGCAACAAA TTTTAGAATT TG -             #GTCAAGTG   1620                                                                  - - CGTCGCGGAT TGCTTGGTAT TAAAGGTGGC GAACTCAATG CTGATTTAGC CA -             #AAGCCTTT   1680                                                                  - - AATGTAAGCG CGCAACAAGG CGCATTTGTA AGTGAAGTTT TACCGAAATC TG -             #CTGCTGAA   1740                                                                  - - AAAGCAGGAC TTAAAGCGGG CGATATTATC ACGGCGATGA ACGGTCAAAA AA -             #TCTCAAGT   1800                                                                  - - TTCGCTGAAA TTCGTGCAAA AATCGCAACC ACTGGTGCAG GCAAAGAGAT TA -             #GCTTGACT   1860                                                                  - - TACTTACGTG ATGGCAAATC CCACGACGTT AAAATGAAAT TACAAGCGGA TG -             #ATAGTAGC   1920                                                                  - - CAACTTTCCT CAAAAACTGA GTTGCCTGCA TTAGATGGTG CAACATTGAA AG -             #ACTACGAT   1980                                                                  - - GCTAAAGGCG TTAAAGGAAT TGAAATCACA AAAATTCAAC CTAATTCGCT GG -             #CTGCACAA   2040                                                                  - - CGTGGTTTAA AATCGGGCGA TATTATTATT GGTATTAATC GTCAAATGAT CG -             #AAAACATT   2100                                                                  - - CGTGAATTAA ATAAAGTGCT TGAAACTGAA CCGTCAGCAG TTGCACTTAA TA -             #TTTTACGA   2160                                                                  - - GGTGACAGTA ATTTCTATTT ATTAGTGCAA TAATCTGCTT GATATATTTA AG -             #AAAAAAGT   2220                                                                  - - CCGATCACAA TGATCGGGCT TCTTTTTATG CAGCAATCGT TCTTAACAAA TC -             #CACCACAA   2280                                                                  - - ATTCTAACCG CACTTTGTTA TCAGATAAAT CTTTCATGAA CTTAAATTTT AA -             #TGGGCCAT   2340                                                                  - - CAAATCGATA CACAATAGGT TCTTTTTGAA TTAATTGAAT AAATTTATCT GG -             #ATTCACTT   2400                                                                  - - GTGCTTTTGC TGAAAACTCA ATAAAACCGC CTTGTGTTCC TGCATCAATT CG -             #CACAACTT   2460                                                                  - - TCAACGGCTC AACCAACAAA CGCAATTCTG CAATTTGCAG TAAATTTTTT GT -             #TGCATCAG   2520                                                                  - - GCAATAATCC GAATCGATCT ATTAACTCAA CTTTTAATTC ATCTAATTCT GC -             #TTTACTCT   2580                                                                  - - CTGCTGCAGC AATGCGTTTA TAAAAGGATA AACGCATATT CACGTCTCCT AG -             #ATAATCAT   2640                                                                  - - CAGGCAGTAA AGCAGGCACA CGCAATTCAA TATCCGCTTG TTGTTGCGTC AA -             #TTCTTCTA   2700                                                                  - - ATGATGGTTC ACGCCCTTCT TTTAACGCTT TAACCGCTGC ATCCAATAAT TC -             #CATATAAA   2760                                                                  - - GCGAAAAACC GATGCTTTCA ATTTGTCCAC TTTGTTCGTT TCCAAGTAAT TC -             #GCCGGCAC   2820                                                                  - - CACGAATCTC TAAATCGTGG GTTGCCAAGA TAAAACCAGC CCCAAGATTA TC -             #AAGATTTT   2880                                                                  - - CCAAGGCATC TAGA              - #                  - #                       - #   2894                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 463 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Met Lys Lys Thr Arg Phe Val Leu Asn Ser Il - #e Ala Leu Gly Leu Ser       1               5   - #                10  - #                15                - - Val Leu Ser Thr Ser Phe Val Ala Gln Ala Th - #r Leu Pro Ser Phe Val                   20      - #            25      - #            30                    - - Ser Glu Gln Asn Ser Leu Ala Pro Met Leu Gl - #u Lys Val Gln Pro Ala               35          - #        40          - #        45                        - - Val Val Thr Leu Ser Val Glu Gly Lys Ala Ly - #s Val Asp Ser Arg Ser           50              - #    55              - #    60                            - - Pro Phe Leu Asp Asp Ile Pro Glu Glu Phe Ly - #s Phe Phe Phe Gly Asp       65                  - #70                  - #75                  - #80         - - Arg Phe Ala Glu Gln Phe Gly Gly Arg Gly Gl - #u Ser Lys Arg Asn Phe                       85  - #                90  - #                95                - - Arg Gly Leu Gly Ser Gly Val Ile Ile Asn Al - #a Ser Lys Gly Tyr Val                   100      - #           105      - #           110                   - - Leu Thr Asn Asn His Val Ile Asp Glu Ala As - #p Lys Ile Thr Val Gln               115          - #       120          - #       125                       - - Leu Gln Asp Gly Arg Glu Phe Lys Ala Lys Le - #u Val Gly Lys Asp Glu           130              - #   135              - #   140                           - - Leu Ser Asp Ile Ala Leu Val Gln Leu Glu Ly - #s Pro Ser Asn Leu Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Glu Ile Lys Phe Ala Asp Ser Asp Lys Leu Ar - #g Val Gly Asp Phe         Thr                                                                                              165  - #               170  - #               175              - - Val Ala Ile Gly Asn Pro Phe Gly Leu Gly Gl - #n Thr Val Thr Ser Gly                   180      - #           185      - #           190                   - - Ile Val Ser Ala Leu Gly Arg Ser Thr Gly Se - #r Asp Ser Gly Thr Tyr               195          - #       200          - #       205                       - - Glu Asn Tyr Ile Gln Thr Asp Ala Ala Val As - #n Arg Gly Asn Ser Gly           210              - #   215              - #   220                           - - Gly Ala Leu Val Asn Leu Asn Gly Glu Leu Il - #e Gly Ile Asn Thr Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ile Ile Ser Pro Ser Gly Gly Asn Ala Gly Il - #e Ala Phe Ala Ile         Pro                                                                                              245  - #               250  - #               255              - - Ser Asn Gln Ala Ser Asn Leu Val Gln Gln Il - #e Leu Glu Phe Gly Gln                   260      - #           265      - #           270                   - - Val Arg Arg Gly Leu Leu Gly Ile Lys Gly Gl - #y Glu Leu Asn Ala Asp               275          - #       280          - #       285                       - - Leu Ala Lys Ala Phe Asn Val Ser Ala Gln Gl - #n Gly Ala Phe Val Ser           290              - #   295              - #   300                           - - Glu Val Leu Pro Lys Ser Ala Ala Glu Lys Al - #a Gly Leu Lys Ala Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Asp Ile Ile Thr Ala Met Asn Gly Gln Lys Il - #e Ser Ser Phe Ala         Glu                                                                                              325  - #               330  - #               335              - - Ile Arg Ala Lys Ile Ala Thr Thr Gly Ala Gl - #y Lys Glu Ile Ser Leu                   340      - #           345      - #           350                   - - Thr Tyr Leu Arg Asp Gly Lys Ser His Asp Va - #l Lys Met Lys Leu Gln               355          - #       360          - #       365                       - - Ala Asp Asp Ser Ser Gln Leu Ser Ser Lys Th - #r Glu Leu Pro Ala Leu           370              - #   375              - #   380                           - - Asp Gly Ala Thr Leu Lys Asp Tyr Asp Ala Ly - #s Gly Val Lys Gly Ile       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Glu Ile Thr Lys Ile Gln Pro Asn Ser Leu Al - #a Ala Gln Arg Gly         Leu                                                                                              405  - #               410  - #               415              - - Lys Ser Gly Asp Ile Ile Ile Gly Ile Asn Ar - #g Gln Met Ile Glu Asn                   420      - #           425      - #           430                   - - Ile Arg Glu Leu Asn Lys Val Leu Glu Thr Gl - #u Pro Ser Ala Val Ala               435          - #       440          - #       445                       - - Leu Asn Ile Leu Arg Gly Asp Ser Asn Phe Ty - #r Leu Leu Val Gln               450              - #   455              - #   460                           - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 41 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - ATGAAAAAAA CACGTTTTGT ATTAAATAGT ATTGCACTTG G    - #                       - #   41                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - Met Lys Lys Thr Arg Phe Val Leu Asn Ser Il - #e Ala Leu Gly Leu Ser       1               5   - #                10  - #                15                - - Val Leu Ser Thr Ser Phe Val Ala Gln Ala Th - #r Leu Pro Ser Phe Val                   20      - #            25      - #            30                    - - Ser Glu Gln Asn Ser                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 472 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - Met Lys Lys Thr Thr Leu Ala Leu Ser Arg Le - #u Ala Leu Ser Leu Ser       1               5   - #                10  - #                15                - - Leu Ala Leu Ser Pro Leu Ser Ala Thr Ala Al - #a Glu Thr Ser Ser Ala                   20      - #            25      - #            30                    - - Thr Thr Ala Gln Gln Met Pro Ser Leu Ala Pr - #o Met Leu Glu Lys Val               35          - #        40          - #        45                        - - Met Pro Ser Val Val Ser Ile Asn Val Glu Gl - #y Ser Thr Thr Val Asn           50              - #    55              - #    60                            - - Thr Pro Arg Met Pro Arg Asn Phe Gln Gln Ph - #e Phe Gly Asp Asp Ser       65                  - #70                  - #75                  - #80         - - Pro Phe Cys Gln Glu Gly Ser Pro Phe Gln Se - #r Ser Pro Phe Cys Gln                       85  - #                90  - #                95                - - Gly Gly Gln Gly Gly Asn Gly Gly Gly Gln Gl - #n Gln Lys Phe Met Ala                   100      - #           105      - #           110                   - - Leu Gly Ser Gly Val Ile Ile Asp Ala Asp Ly - #s Gly Tyr Val Val Thr               115          - #       120          - #       125                       - - Asn Asn His Val Val Asp Asn Ala Thr Val Il - #e Lys Val Gln Leu Ser           130              - #   135              - #   140                           - - Asp Gly Arg Lys Phe Asp Ala Lys Met Val Gl - #y Lys Asp Pro Arg Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Ile Ala Leu Ile Gln Ile Gln Asn Pro Ly - #s Asn Leu Thr Ala         Ile                                                                                              165  - #               170  - #               175              - - Lys Met Ala Asp Ser Asp Ala Leu Arg Val Gl - #y Asp Tyr Thr Val Gly                   180      - #           185      - #           190                   - - Ile Gly Asn Pro Phe Gly Leu Gly Glu Thr Va - #l Thr Ser Gly Ile Val               195          - #       200          - #       205                       - - Ser Ala Leu Gly Arg Ser Gly Leu Asn Ala Gl - #u Asn Tyr Glu Asn Phe           210              - #   215              - #   220                           - - Ile Gln Thr Asp Ala Ala Ile Asn Arg Gly As - #n Ser Gly Gly Ala Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Asn Leu Asn Gly Glu Leu Ile Gly Ile As - #n Thr Ala Ile Leu         Ala                                                                                              245  - #               250  - #               255              - - Pro Asp Gly Gly Asn Ile Gly Ile Gly Phe Al - #a Ile Pro Ser Asn Met                   260      - #           265      - #           270                   - - Val Lys Asn Leu Thr Ser Gln Met Val Glu Ty - #r Gly Gln Val Lys Arg               275          - #       280          - #       285                       - - Gly Glu Leu Gly Ile Met Gly Thr Glu Leu As - #n Ser Glu Leu Ala Lys           290              - #   295              - #   300                           - - Ala Met Lys Val Asp Ala Gln Arg Gly Ala Ph - #e Val Ser Gln Val Leu       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Pro Asn Ser Ser Ala Ala Lys Ala Gly Ile Ly - #s Ala Gly Asp Val         Ile                                                                                              325  - #               330  - #               335              - - Thr Ser Leu Asn Gly Lys Pro Ile Ser Ser Ph - #e Ala Ala Leu Arg Ala                   340      - #           345      - #           350                   - - Gln Val Gly Thr Met Pro Val Gly Ser Lys Le - #u Thr Leu Gly Leu Leu               355          - #       360          - #       365                       - - Arg Asp Gly Lys Gln Val Asn Val Asn Leu Gl - #u Leu Gln Gln Ser Ser           370              - #   375              - #   380                           - - Gln Asn Gln Val Asp Ser Ser Ser Ile Phe As - #n Gly Ile Glu Gly Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Glu Met Ser Asn Lys Gly Lys Asp Gln Gly Va - #l Val Val Asn Asn         Val                                                                                              405  - #               410  - #               415              - - Lys Thr Gly Thr Pro Ala Ala Gln Ile Gly Le - #u Lys Lys Gly Asp Val                   420      - #           425      - #           430                   - - Ile Ile Gly Ala Asn Gln Ile Ala Val Lys As - #n Ile Ala Glu Ile Arg               435          - #       440          - #       445                       - - Lys Val Leu Asp Ser Lys Pro Ser Val Leu Al - #a Leu Asn Ile Gln Arg           450              - #   455              - #   460                           - - Gly Asp Arg His Leu Pro Val Asn                                           465                 4 - #70                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 475 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - Met Lys Lys Thr Thr Leu Ala Met Ser Ala Le - #u Ala Leu Ser Leu Gly       1               5   - #                10  - #                15                - - Leu Ala Leu Ser Pro Leu Ser Ala Thr Ala Al - #a Glu Thr Ser Ser Ser                   20      - #            25      - #            30                    - - Ala Met Thr Ala Gln Gln Met Pro Ser Leu Al - #a Pro Met Leu Glu Lys               35          - #        40          - #        45                        - - Val Met Pro Ser Val Val Ser Ile Asn Val Gl - #u Gly Ser Thr Thr Val           50              - #    55              - #    60                            - - Asn Thr Pro Arg Met Pro Arg Asn Phe Gln Gl - #n Phe Phe Gly Asp Asp       65                  - #70                  - #75                  - #80         - - Ser Pro Phe Cys Gln Asp Gly Ser Pro Phe Gl - #n Asn Ser Pro Phe Cys                       85  - #                90  - #                95                - - Gln Gly Gly Gly Asn Gly Gly Asn Gly Gly Gl - #n Gln Gln Lys Phe Met                   100      - #           105      - #           110                   - - Ala Leu Gly Ser Gly Val Ile Ile Asp Ala As - #p Lys Gly Tyr Val Val               115          - #       120          - #       125                       - - Thr Asn Asn His Val Val Asp Asn Ala Ser Va - #l Ile Lys Val Gln Leu           130              - #   135              - #   140                           - - Ser Asp Gly Arg Lys Phe Asp Ala Lys Val Va - #l Gly Lys Asp Pro Arg       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Asp Ile Ala Leu Ile Gln Ile Gln Asn Pr - #o Lys Asn Leu Thr         Ala                                                                                              165  - #               170  - #               175              - - Ile Lys Leu Ala Asp Ser Asp Ala Leu Arg Va - #l Gly Asp Tyr Thr Val                   180      - #           185      - #           190                   - - Ala Ile Gly Asn Pro Phe Gly Leu Gly Glu Th - #r Val Thr Ser Gly Ile               195          - #       200          - #       205                       - - Val Ser Ala Leu Gly Arg Ser Gly Leu Asn Va - #l Glu Asn Tyr Glu Asn           210              - #   215              - #   220                           - - Phe Ile Gln Thr Asp Ala Ala Ile Asn Arg Gl - #y Asn Ser Gly Gly Ala       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Val Asn Leu Asn Gly Glu Leu Ile Gly Il - #e Asn Thr Ala Ile         Leu                                                                                              245  - #               250  - #               255              - - Ala Pro Asp Gly Gly Asn Ile Gly Ile Gly Ph - #e Ala Ile Pro Ser Asn                   260      - #           265      - #           270                   - - Met Val Lys Asn Leu Thr Ser Gln Met Val Gl - #u Tyr Gly Gln Val Arg               275          - #       280          - #       285                       - - Arg Gly Glu Leu Gly Ile Met Gly Thr Glu Le - #u Asn Ser Glu Leu Ala           290              - #   295              - #   300                           - - Lys Ala Met Lys Val Asp Ala Gln Arg Gly Al - #a Phe Val Ser Gln Val       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Met Pro Asn Ser Ser Ala Ala Lys Ala Gly Il - #e Lys Ala Gly Asp         Val                                                                                              325  - #               330  - #               335              - - Ile Thr Ser Leu Asn Gly Lys Pro Ile Ser Se - #r Phe Ala Ala Leu Arg                   340      - #           345      - #           350                   - - Ala Gln Val Gly Thr Met Pro Val Gly Ser Ly - #s Ile Ser Leu Gly Leu               355          - #       360          - #       365                       - - Leu Arg Glu Gly Lys Ala Ile Thr Val Asn Le - #u Glu Leu Gln Gln Ser           370              - #   375              - #   380                           - - Ser Gln Ser Gln Val Asp Ser Ser Thr Ile Ph - #e Ser Gly Ile Glu Gly       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ala Glu Met Ser Asn Lys Gly Gln Asp Lys Gl - #y Val Val Val Ser         Ser                                                                                              405  - #               410  - #               415              - - Val Lys Ala Asn Ser Pro Ala Ala Gln Ile Gl - #y Leu Lys Lys Gly Asp                   420      - #           425      - #           430                   - - Val Ile Ile Gly Ala Asn Gln Ile Pro Val Ly - #s Asn Ile Ala Glu Ile               435          - #       440          - #       445                       - - Arg Lys Ile Leu Asp Ser Lys Pro Ser Val Le - #u Ala Leu Asn Ile Gln           450              - #   455              - #   460                           - - Arg Gly Asp Ser Ser Ile Tyr Leu Leu Met Gl - #n                           465                 4 - #70                 4 - #75                             - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 228 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - Ile Val Gly Gly Tyr Lys Cys Glu Lys Asn Se - #r Gln Pro Trp Gln Val       1               5   - #                10  - #                15                - - Ala Val Ile Asn Glu Tyr Leu Cys Gly Gly Va - #l Leu Ile Asp Pro Ser                   20      - #            25      - #            30                    - - Trp Val Ile Thr Ala Ala His Cys Tyr Ser As - #n Asn Tyr Gln Val Leu               35          - #        40          - #        45                        - - Leu Gly Arg Asn Asn Leu Phe Lys Asp Glu Pr - #o Phe Ala Gln Arg Arg           50              - #    55              - #    60                            - - Leu Val Pro Gln Ser Phe Arg His Pro Asp Ty - #r Ile Pro Leu Ile Pro       65                  - #70                  - #75                  - #80         - - Val His Asp His Ser Asn Asp Leu Met Leu Le - #u His Leu Ser Glu Pro                       85  - #                90  - #                95                - - Ala Asp Ile Thr Gly Gly Val Lys Val Ile As - #p Leu Pro Thr Lys Glu                   100      - #           105      - #           110                   - - Pro Lys Val Gly Ser Thr Cys Leu Ala Ser Gl - #y Trp Gly Ser Thr Asn               115          - #       120          - #       125                       - - Pro Ser Glu Met Val Val Ser His Asp Leu Gl - #n Cys Val Asn Ile His           130              - #   135              - #   140                           - - Leu Leu Ser Asn Glu Lys Cys Ile Glu Thr Ty - #r Lys Asp Asn Val Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Val Met Leu Cys Ala Gly Glu Met Glu Gl - #y Gly Lys Asp Thr         Cys                                                                                              165  - #               170  - #               175              - - Ala Gly Asp Ser Gly Gly Pro Leu Ile Cys As - #p Gly Val Leu Gln Gly                   180      - #           185      - #           190                   - - Ile Thr Ser Gly Gly Ala Thr Pro Cys Ala Ly - #s Pro Lys Thr Pro Ala               195          - #       200          - #       205                       - - Ile Tyr Ala Lys Leu Ile Lys Phe Thr Ser Tr - #p Ile Lys Lys Val Met           210              - #   215              - #   220                           - - Lys Glu Asn Pro                                                           225                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 232 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - Ile Ile Gly Gly Arg Glu Cys Glu Lys Asn Se - #r His Pro Trp Gln Val       1               5   - #                10  - #                15                - - Ala Ile Tyr His Tyr Ser Ser Phe Gln Cys Gl - #y Gly Val Leu Val Asn                   20      - #            25      - #            30                    - - Pro Lys Trp Val Leu Thr Ala Ala His Cys Ly - #s Asn Asp Asn Tyr Glu               35          - #        40          - #        45                        - - Val Trp Leu Gly Arg His Asn Leu Phe Glu As - #n Glu Asn Thr Ala Gln           50              - #    55              - #    60                            - - Phe Phe Gly Val Thr Ala Asp Phe Pro His Pr - #o Gly Phe Asn Leu Ser       65                  - #70                  - #75                  - #80         - - Ala Asp Gly Lys Asp Tyr Ser His Asp Leu Me - #t Leu Leu Arg Leu Gln                       85  - #                90  - #                95                - - Ser Pro Ala Lys Ile Thr Asp Ala Val Lys Va - #l Leu Glu Leu Pro Thr                   100      - #           105      - #           110                   - - Gln Glu Pro Glu Leu Gly Ser Thr Cys Glu Al - #a Ser Gly Trp Gly Ser               115          - #       120          - #       125                       - - Ile Glu Pro Gly Pro Asp Asp Phe Glu Phe Pr - #o Asp Glu Ile Gln Cys           130              - #   135              - #   140                           - - Val Gln Leu Thr Leu Leu Gln Asn Thr Phe Cy - #s Ala Asp Ala His Pro       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Lys Val Thr Glu Ser Met Leu Cys Ala Gl - #y Tyr Leu Pro Gly         Gly                                                                                              165  - #               170  - #               175              - - Lys Asp Thr Cys Met Gly Asp Ser Gly Gly Pr - #o Leu Ile Cys Asn Gly                   180      - #           185      - #           190                   - - Met Trp Gln Gly Ile Thr Ser Trp Gly His Th - #r Pro Cys Gly Ser Ala               195          - #       200          - #       205                       - - Asn Lys Pro Ser Ile Tyr Thr Lys Leu Ile Ph - #e Tyr Leu Asp Trp Ile           210              - #   215              - #   220                           - - Asp Asp Thr Ile Thr Glu Asn Pro                                           225                 2 - #30                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 223 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - Ile Val Gly Gly Tyr Thr Cys Gly Ala Asn Th - #r Val Pro Tyr Gln Val       1               5   - #                10  - #                15                - - Ser Leu Asn Ser Gly Tyr His Phe Cys Gly Gl - #y Ser Leu Ile Asn Ser                   20      - #            25      - #            30                    - - Gln Trp Val Val Ser Ala Ala His Cys Tyr Ly - #s Ser Gly Ile Gln Val               35          - #        40          - #        45                        - - Arg Leu Gly Glu Asp Asn Ile Asn Val Val Gl - #u Gly Asn Glu Gln Phe           50              - #    55              - #    60                            - - Ile Ser Ala Ser Lys Ser Ile Val His Pro Se - #r Tyr Asn Ser Asn Thr       65                  - #70                  - #75                  - #80         - - Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Ly - #s Ser Ala Ala Ser Leu                       85  - #                90  - #                95                - - Asn Ser Arg Val Ala Ser Ile Ser Leu Pro Th - #r Ser Cys Ala Ser Ala                   100      - #           105      - #           110                   - - Gly Thr Gln Cys Leu Ile Ser Gly Trp Gly As - #n Thr Lys Ser Ser Gly               115          - #       120          - #       125                       - - Thr Ser Tyr Pro Asp Val Leu Lys Cys Leu Ly - #s Ala Pro Ile Leu Ser           130              - #   135              - #   140                           - - Asp Ser Ser Cys Lys Ser Ala Tyr Pro Gly Gl - #n Ile Thr Ser Asn Met       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Phe Cys Ala Gly Tyr Leu Glu Gly Gly Lys As - #p Ser Cys Gln Gly         Asp                                                                                              165  - #               170  - #               175              - - Ser Gly Gly Pro Val Val Cys Ser Gly Lys Le - #u Gln Gly Ile Val Ser                   180      - #           185      - #           190                   - - Trp Gly Ser Gly Cys Ala Gln Lys Asn Lys Pr - #o Gly Val Tyr Thr Lys               195          - #       200          - #       205                       - - Val Cys Asn Tyr Val Ser Trp Ile Lys Gln Th - #r Ile Ala Ser Asn               210              - #   215              - #   220                           - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 228 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - Ile Val Asn Gly Glu Glu Ala Val Pro Gly Se - #r Trp Pro Trp Gln Val       1               5   - #                10  - #                15                - - Ser Leu Gln Asp Lys Thr Gly Phe His Phe Cy - #s Gly Gly Ser Leu Ile                   20      - #            25      - #            30                    - - Asn Glu Asn Trp Val Val Thr Ala Ala His Cy - #s Gly Val Thr Thr Ser               35          - #        40          - #        45                        - - Asp Val Val Val Ala Gly Glu Phe Asp Gln Gl - #y Ser Ser Ser Glu Lys           50              - #    55              - #    60                            - - Ile Gln Lys Leu Lys Ile Ala Lys Val Phe Ly - #s Asn Ser Lys Tyr Asn       65                  - #70                  - #75                  - #80         - - Ser Leu Thr Ile Asn Asn Asp Ile Thr Leu Le - #u Lys Leu Ser Thr Ala                       85  - #                90  - #                95                - - Ala Ser Phe Ser Gln Thr Val Ser Ala Val Cy - #s Leu Pro Ser Ala Ser                   100      - #           105      - #           110                   - - Asp Asp Phe Ala Ala Gly Thr Thr Cys Val Th - #r Thr Gly Trp Gly Leu               115          - #       120          - #       125                       - - Thr Arg Tyr Ala Asn Thr Pro Asp Arg Leu Gl - #n Gln Ala Ser Leu Pro           130              - #   135              - #   140                           - - Leu Leu Ser Asn Thr Asn Cys Lys Lys Tyr Tr - #p Gly Thr Lys Ile Lys       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Ala Met Ile Cys Ala Gly Ala Ser Gly Va - #l Ser Ser Cys Met         Gly                                                                                              165  - #               170  - #               175              - - Asp Ser Gly Gly Pro Leu Val Cys Lys Lys As - #n Gly Ala Trp Thr Leu                   180      - #           185      - #           190                   - - Val Gly Ile Val Ser Trp Gly Ser Ser Thr Cy - #s Ser Thr Ser Thr Pro               195          - #       200          - #       205                       - - Gly Val Tyr Ala Arg Val Thr Ala Leu Val As - #n Trp Val Gln Gln Thr           210              - #   215              - #   220                           - - Leu Ala Ala Asn                                                           225                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 240 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - Val Val Gly Gly Thr Glu Ala Gln Arg Asn Se - #r Trp Pro Ser Gln Ile       1               5   - #                10  - #                15                - - Ser Leu Gln Tyr Arg Ser Gly Ser Ser Trp Al - #a His Thr Cys Gly Gly                   20      - #            25      - #            30                    - - Thr Leu Ile Arg Gln Asn Trp Val Met Thr Al - #a Ala His Cys Val Asp               35          - #        40          - #        45                        - - Arg Glu Leu Thr Phe Arg Val Val Val Gly Gl - #u His Asn Leu Asn Gln           50              - #    55              - #    60                            - - Asn Asn Gly Thr Glu Gln Tyr Val Gly Val Gl - #n Lys Ile Val Val His       65                  - #70                  - #75                  - #80         - - Pro Tyr Trp Asn Thr Asp Asp Val Ala Ala Gl - #y Tyr Asp Ile Ala Leu                       85  - #                90  - #                95                - - Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Se - #r Tyr Val Gln Leu Gly                   100      - #           105      - #           110                   - - Val Leu Pro Arg Ala Gly Thr Ile Leu Ala As - #n Asn Ser Pro Cys Tyr               115          - #       120          - #       125                       - - Ile Thr Gly Trp Gly Leu Thr Arg Thr Asn Gl - #y Gln Leu Ala Gln Thr           130              - #   135              - #   140                           - - Leu Gln Gln Ala Tyr Leu Pro Thr Val Asp Ty - #r Ala Ile Cys Ser Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Se - #r Met Val Cys Ala         Gly                                                                                              165  - #               170  - #               175              - - Gly Asp Gly Val Arg Ser Gly Cys Gln Gly As - #p Ser Gly Gly Pro Leu                   180      - #           185      - #           190                   - - His Cys Leu Val Asn Gly Gln Tyr Ala Val Hi - #s Gly Val Thr Ser Phe               195          - #       200          - #       205                       - - Val Ser Arg Leu Gly Cys Asn Val Thr Arg Ly - #s Pro Thr Val Phe Thr           210              - #   215              - #   220                           - - Arg Val Ser Ala Tyr Ile Ser Trp Ile Asn As - #n Val Ile Ala Ser Asn       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 224 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - Ile Ile Gly Gly Val Glu Ser Ile Pro His Se - #r Arg Pro Tyr Met         Ala                                                                              1               5   - #                10  - #                15               - - His Leu Asp Ile Val Thr Glu Lys Gly Leu Ar - #g Val Ile Cys Gly Gly                   20      - #            25      - #            30                    - - Phe Leu Ile Ser Arg Gln Phe Val Leu Thr Al - #a Ala His Cys Lys Gly               35          - #        40          - #        45                        - - Arg Glu Ile Thr Val Ile Leu Gly Ala His As - #p Val Arg Lys Arg Glu           50              - #    55              - #    60                            - - Ser Thr Gln Gln Lys Ile Lys Val Glu Lys Gl - #n Ile Ile His Glu Ser       65                  - #70                  - #75                  - #80         - - Tyr Asn Ser Val Pro Asn Leu His Asp Ile Me - #t Leu Leu Lys Leu Glu                       85  - #                90  - #                95                - - Lys Lys Val Glu Leu Thr Pro Ala Val Asn Va - #l Val Pro Leu Pro Ser                   100      - #           105      - #           110                   - - Pro Ser Asp Phe Ile His Pro Gly Ala Met Cy - #s Trp Ala Ala Gly Trp               115          - #       120          - #       125                       - - Gly Lys Thr Gly Val Arg Asp Pro Thr Ser Ty - #r Thr Leu Arg Glu Val           130              - #   135              - #   140                           - - Glu Leu Arg Ile Met Asp Glu Lys Ala Cys Va - #l Asp Tyr Arg Tyr Tyr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Glu Tyr Lys Phe Gln Val Cys Val Gly Ser Pr - #o Thr Thr Leu Arg         Ala                                                                                              165  - #               170  - #               175              - - Ala Phe Met Gly Asp Ser Gly Gly Pro Leu Le - #u Cys Ala Gly Val Ala                   180      - #           185      - #           190                   - - His Gly Ile Val Ser Tyr Gly His Pro Asp Al - #a Lys Pro Pro Ala Ile               195          - #       200          - #       205                       - - Phe Thr Arg Val Ser Thr Tyr Val Pro Trp Il - #e Asn Ala Val Ile Asn           210              - #   215              - #   220                           - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 223 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - Val Val Gly Gly Thr Arg Ala Ala Gln Gly Gl - #u Phe Pro Phe Met Val       1               5   - #                10  - #                15                - - Arg Leu Ser Met Gly Cys Gly Gly Ala Leu Ty - #r Ala Gln Asp Ile Val                   20      - #            25      - #            30                    - - Leu Thr Ala Ala His Cys Val Ser Gly Ser Gl - #y Asn Asn Thr Ser Ile               35          - #        40          - #        45                        - - Thr Ala Thr Gly Gly Val Val Asp Leu Gln Se - #r Gly Ala Ala Val Lys           50              - #    55              - #    60                            - - Val Arg Ser Thr Lys Val Leu Gln Ala Pro Gl - #y Tyr Asn Gly Thr Gly       65                  - #70                  - #75                  - #80         - - Lys Asp Trp Ala Leu Ile Lys Leu Ala Gln Pr - #o Ile Asn Gln Pro Thr                       85  - #                90  - #                95                - - Leu Lys Ile Ala Thr Thr Thr Ala Tyr Asn Gl - #n Gly Thr Phe Thr Val                   100      - #           105      - #           110                   - - Ala Gly Trp Gly Ala Asn Arg Glu Gly Gly Se - #r Gln Gln Arg Tyr Leu               115          - #       120          - #       125                       - - Leu Lys Ala Asn Val Pro Phe Val Ser Asp Al - #a Ala Cys Arg Ser Ala           130              - #   135              - #   140                           - - Tyr Gly Asn Glu Leu Val Ala Asn Glu Glu Il - #e Cys Ala Gly Tyr Pro       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Thr Gly Gly Val Asp Thr Cys Gln Gly As - #p Ser Gly Gly Pro         Met                                                                                              165  - #               170  - #               175              - - Phe Arg Lys Asp Asn Ala Asp Glu Trp Ile Gl - #n Val Gly Ile Val Ser                   180      - #           185      - #           190                   - - Trp Gly Tyr Gly Cys Ala Arg Pro Gly Tyr Pr - #o Gly Val Tyr Thr Glu               195          - #       200          - #       205                       - - Val Ser Thr Phe Ala Ser Ala Ile Ala Ser Al - #a Ala Arg Thr Leu               210              - #   215              - #   220                           - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 185 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - Ile Ser Gly Gly Asp Ala Ile Tyr Ser Ser Th - #r Gly Arg Cys Ser Leu       1               5   - #                10  - #                15                - - Gly Phe Asn Val Arg Ser Gly Ser Thr Tyr Ty - #r Phe Leu Thr Ala Gly                   20      - #            25      - #            30                    - - His Cys Thr Asp Gly Ala Thr Thr Trp Trp Al - #a Asn Ser Ala Arg Thr               35          - #        40          - #        45                        - - Thr Val Leu Gly Thr Thr Ser Gly Ser Ser Ph - #e Pro Asn Asn Asp Tyr           50              - #    55              - #    60                            - - Gly Ile Val Arg Tyr Thr Asn Thr Thr Ile Pr - #o Lys Asp Gly Thr Val       65                  - #70                  - #75                  - #80         - - Gly Gly Gln Asp Ile Thr Ser Ala Ala Asn Al - #a Thr Val Gly Met Ala                       85  - #                90  - #                95                - - Val Thr Arg Arg Gly Ser Thr Thr Gly Thr Hi - #s Ser Gly Ser Val Thr                   100      - #           105      - #           110                   - - Ala Leu Asn Ala Thr Val Asn Tyr Gly Gly Gl - #y Asp Val Val Tyr Gly               115          - #       120          - #       125                       - - Met Ile Arg Thr Asn Val Cys Ala Glu Pro Gl - #y Asp Ser Gly Gly Pro           130              - #   135              - #   140                           - - Leu Tyr Ser Gly Thr Arg Ala Ile Gly Leu Th - #r Ser Gly Gly Ser Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asn Cys Ser Ser Gly Gly Thr Thr Phe Phe Gl - #n Pro Val Thr Glu         Ala                                                                                              165  - #               170  - #               175              - - Leu Val Ala Tyr Gly Val Ser Val Tyr                                                   180      - #           185                                          - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 181 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - Ile Ala Gly Gly Glu Ala Ile Thr Thr Gly Gl - #y Ser Arg Cys Ser Leu       1               5   - #                10  - #                15                - - Gly Phe Asn Val Ser Val Asn Gly Val Ala Hi - #s Ala Leu Thr Ala Gly                   20      - #            25      - #            30                    - - His Cys Thr Asn Ile Ser Ala Ser Trp Ser Il - #e Gly Thr Arg Thr Gly               35          - #        40          - #        45                        - - Thr Ser Phe Pro Asn Asn Asp Tyr Gly Ile Il - #e Arg His Ser Asn Pro           50              - #    55              - #    60                            - - Ala Ala Ala Asp Gly Arg Val Tyr Leu Tyr As - #n Gly Ser Tyr Gln Asp       65                  - #70                  - #75                  - #80         - - Ile Thr Thr Ala Gly Asn Ala Phe Val Gly Gl - #n Ala Val Gln Arg Ser                       85  - #                90  - #                95                - - Gly Ser Thr Thr Gly Leu Arg Ser Gly Ser Va - #l Thr Gly Leu Asn Ala                   100      - #           105      - #           110                   - - Thr Val Asn Tyr Gly Ser Ser Gly Ile Val Ty - #r Gly Met Ile Gln Thr               115          - #       120          - #       125                       - - Asn Val Cys Ala Gln Pro Gly Asp Ser Gly Gl - #y Ser Leu Phe Ala Gly           130              - #   135              - #   140                           - - Ser Thr Ala Leu Gly Leu Thr Ser Gly Gly Se - #r Gly Asn Cys Arg Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gly Gly Thr Thr Phe Tyr Gln Pro Val Thr Gl - #u Ala Leu Ser Ala         Tyr                                                                                              165  - #               170  - #               175              - - Gly Ala Thr Val Leu                                                                   180                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 198 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - Ala Asn Ile Val Gly Gly Ile Glu Tyr Ser Il - #e Asn Asn Ala Ser Leu       1               5   - #                10  - #                15                - - Cys Ser Val Gly Phe Ser Val Thr Arg Gly Al - #a Thr Lys Gly Phe Val                   20      - #            25      - #            30                    - - Thr Ala Gly His Cys Gly Thr Val Asn Ala Th - #r Ala Arg Ile Gly Gly               35          - #        40          - #        45                        - - Ala Val Val Gly Thr Phe Ala Ala Arg Val Ph - #e Pro Gly Asn Asp Arg           50              - #    55              - #    60                            - - Ala Trp Val Ser Leu Thr Ser Ala Gln Thr Le - #u Leu Pro Arg Val Ala       65                  - #70                  - #75                  - #80         - - Asn Gly Ser Ser Phe Val Thr Val Arg Gly Se - #r Thr Glu Ala Ala Val                       85  - #                90  - #                95                - - Gly Ala Ala Val Cys Arg Ser Gly Arg Thr Th - #r Gly Tyr Gln Cys Gly                   100      - #           105      - #           110                   - - Thr Ile Thr Ala Lys His Val Thr Ala Asn Ty - #r Ala Glu Gly Ala Val               115          - #       120          - #       125                       - - Arg Gly Leu Thr Gln Gly Asn Ala Cys Met Gl - #y Arg Gly Asp Ser Gly           130              - #   135              - #   140                           - - Gly Ser Trp Ile Thr Ser Ala Gly Gln Ala Gl - #n Gly Val Met Ser Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gly Asn Val Gln Ser Asn Gly Asn Asn Cys Gl - #y Ile Pro Ala Ser         Gln                                                                                              165  - #               170  - #               175              - - Arg Ser Ser Leu Phe Glu Arg Leu Gln Pro Il - #e Leu Ser Gln Tyr Gly                   180      - #           185      - #           190                   - - Leu Ser Leu Val Thr Gly                                                           195                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino - #acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - Gly Asn Ser Gly Gly Ala Leu                                               1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino - #acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - Gly Asp Ser Gly Gly Pro Lys                                               1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - Met Lys Lys Thr Arg Phe Val Leu Asn Ser Il - #e Ala Leu Gly               1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - CGCTCCACCA GCATTACCGC GG           - #                  - #                      22                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - ATCAATAACA GCATTATTGG T           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - TAATGCAATT GCTGATAGTT C           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - Lys Phe Phe Phe Gly Asp Arg Phe Ala Glu Gl - #n                           1               5   - #                10                                     __________________________________________________________________________ 

What we claim is:
 1. A method of determining the presence of Haemophilus influenzae Hin47 protein in a sample, comprising the steps of:(a) immunizing a subject with an immunogenic composition comprising an immunoeffective amount of (1) an isolated and purified analog of Haemophilus influenzae Hin47 having a decreased protease activity which is less than about 10% of that of natural Hin47 protein and having substantially the same immunological properties as natural Hin47 protein or (2) an isolated and purified nucleic acid molecule comprising a mutant Haemophilus influenzae Hin47 gene encoding an analog of Haemophilus influenzae Hin47 protein having a decreased protease activity which is less than about 10% of that of natural Hin47 protein and having substantially the immunogenic properties of natural Hin47 protein to produce antibodies specifically reactive therewith, (b) contacting the sample with the antibodies to produce complexes comprising any Hin47 protein present in the sample and said Hin47 protein specific antibodies; and (c) determining production of the complexes.
 2. A diagnostic kit for determining the presence of antibodies in a sample specifically reactive with Haemophilus influenzae Hin47 protein, comprising:(a) an isolated and purified analog of Haemphilus influenzae Hin47 having a decreased protease activity which is less than about 10% of that of natural Hin47 protein and having substantially the same immunological properties as natural Hin47 protein; (b) means for contacting the analog with the sample to produce complexes comprising the analog and any said antibodies present in the sample; and (c) means for determining production of the complexes.
 3. A method of determining the presence of antibodies specifically reactive with Haemophilus influenzae Hin47 protein in a sample, comprising the steps of:(a) contacting the sample with an isolated and purified analog of Haemphilus influenzae Hin47 having a decreased protease activity which is less than about 10% of that of natural Hin47 protein and having substantially the same immunological properties as natural Hin47 protein to produce complexes comprising the Hin47 analog and any said antibodies present in the sample specifically reactive therewith; and (b) determining production of the complexes. 